Gamma-1 and gamma-3 anti-human CD23 monoclonal antibodies and use thereof as therapeutics

ABSTRACT

Methods for inhibiting production of IgE in a human subject with an IgE-mediated allergic disorder by administering anti-CD23 antibodies.

RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 09/292,053,filed Apr. 14, 1999, which is a divisional of U.S. patent applicationSer. No. 08/803,085, filed Feb. 20, 1997, now U.S. Pat. No. 6,011,138.

FIELD OF THE INVENTION

The present invention relates to monoclonal antibodies containing humangamma-1 constant domains which specifically bind human CD23, the lowaffinity receptor for IgE (FceRII/CD23) and their usage as therapeuticagents.

BACKGROUND OF THE INVENTION

IgE is a member of the immunoglobulin family that mediates allergicresponses such as asthma, food allergies, type 1 hypersensitivity andthe familiar sinus inflammation allergic rhinitis and conjunctivitis,and as a result, causes widespread suffering throughout the generalpopulation. IgE is secreted by, and expressed on the surface of,B-cells. IgE synthesized by B-cells can be anchored in the B-cellmembrane by a short transmembrane domain linked to the mature IgEsequence. Membrane and secreted versions of IgE are formed in the samecell by differential splicing of the IgE RNA transcript.

IgE also can be bound to B-cells (and T cells, monocytes, Langerhanscells, follicular dendritic cells, natural killer cells, eosinophils andplatelets) through its Fc region to a low affinity IgE receptor (FceRII,hereafter “FCEL”, and to mast cells and basophils through its Fc regionto a high affinity IgE receptor FceRI, (hereinafter “FCEH”). The lowaffinity IgE receptor is generally referred to in the literature asCD23.

Upon exposure of a mammal to an allergen, antigen presenting cellsprocess the antigen for presentation to helper T cells. These helper Tcells secrete cytokines such as IL-4 which assist B-cells to undergoclonal amplification and secrete more allergen-specific IgE. This newlysynthesized IgE in turn is released into the circulation where it bindsto mast cells and basophils through the high affinity receptor on theircell surface. Such mast cells and basophils are thereby sensitized tothe specific allergen. The next exposure to the same allergen causesbinding to specific IgE on the surface of mast cells, and basophils,thereby cross-linking the FceRI on these cells and thus activating theirrelease of histamine and other factors which are responsible forclinical hypersensitivity and anaphylaxis.

The art has reported antibodies capable of binding to FCEL (CD23)-boundIgE but not IgE bound to FCEH (see, for example, WO 89/00138 and U.S.Pat. No. 4,940,782). These antibodies are disclosed to be clinicallyadvantageous because they bind to IgE which is bound to the low affinityreceptor (FCEL) or to circulating IgE's, but do not bind to IgE bound tothe high affinity receptor (FCEH). Therefore, these antibodies will notactivate mast cells or basophils.

Moreover, anti-CD23 antibodies have been reported to have potential astherapeutics, e.g., for the treatment of allergic disorders,inflammatory diseases, and autoimmune diseases. For example, Bonnefoy etal., WO 9612741, report that ligands which bind CD23, e.g., monoclonalantibodies, are useful in the treatment or prophylaxis of inflammatory,autoimmune and allergic diseases.

The usage of monoclonal antibodies to CD23, as both IgE agonists andantagonists has been reported. IgE antagonists have been reported tohave potential utility in treatment of conditions or diseases whereinIgE suppression is therapeutically desirable, e.g., allergic conditionssuch as allergic rhinitis and conjuntivitis, atopic dermatitis andasthma. For example, Bonnefoy et al., WO 8707302 (1987), reportmonoclonal antibodies to human CD23, which are assertedly useful forassaying the presence of IgE receptors on cell types and as therapeuticsin diseases wherein modulation of IgE is therapeutically desirable.

In part because of their potential as therapeutics and diagnostics, manygroups have reported the generation of monoclonal antibodies to CD23.See, e.g., Rector et al., Immunol., 55:481-488 (1985); Suemura et al.,J. Immunol., 137:1214-1220 (1986); Noro et al., J. Immunol.,137:1258-1263 (1986); Bonnefoy et al., J. Immunol., 138:2170-2178(1987); Flores-Romo et al., Science, 261:1038-1046 (1993); Sherr et al.;J. Immunol., 142:481-489 (1989); and Pene et al., Proc. Natl. Acad.Sci., USA, 85:6880-6884 (1988). Moreover, as discussed supra, the usageof such antibodies specifically to inhibit IgE production in systemswhere IgE synthesis is cytokine (IL-4) induced has also been reported.(Flores-Romo et al (Id.); Sherr et al. (Id.); Bonnefoy et al. (WO8707302); Bonnefoy et al. (WO 8707302); Bonnefoy et al. (WO 9612741));Bonnefoy et al., Eur. J. Immunol 20:139-144 (1990); Sarfati et al., J.Immunol 141:2195-2199 (1988) and Wakai et al., Hybridoma 12:25-43(1993). Also, Flores-Romo et al. (Id.) disclose that Fabs prepared fromanti-CD23 antibodies inhibit antigen-specific induced IgE responses invivo in the rat. However, notwithstanding what has been reported, themechanism by which anti-CD23 antibodies modulate IgE expression and inparticular, the manner by which they block IL-4 induced IgE productionremains unclear.

It has been suggested that anti-CD23 antibodies inhibit IgE productionby signaling through CD23 present on the surface of IgE secreting Bcells. It has been proposed that the function of CD23, which isupregulated on IgE secreting B cells, is feedback inhibition of IgEproduction (Yu, et al. Nature 369, 753-756 (1994)). This has beentheorized because mice in which the CD23 gene has been removed haveincreased and sustained IgE production compared to controls (Yu, etal.). In addition, it has been reported that binding to CD23 by IgEcomplexes or by a monoclonal antibody to anti-CD23 suppresses ongoingIgE synthesis by a lymphoblastoid cell line that constitutively secretesIgE (Sherr et al. (Id.)). It appears that this is due to down regulationof the messenger RNA for the secreted IgE heavy chain in this cell(Saxon et al., J. Immunol., 147:4000-4006 (1991)). However, the exactmechanism by which IgE expression is inhibited has yet to be explainedin systems in which IgE secretion is IL-4 induced.

It has also been reported that crosslinking of Fc gamma RII with surfaceIg (B cell receptor) on B cells leads to down regulation of Igexpression. (D'Ambrosia et al., Science, 268:293-297 (1995).) A similarmechanism can be proposed for B cells secreting IgE which also have cellsurface CD23 and Fc gamma RII. An anti-human CD23 antibody bound to acell by antigen (CD23) and also bound to Fc gamma RII through Fcinteractions could transmit a signal to suppress IgE secretion throughFc gamma RII.

Mechanisms involved in IgE inhibition by anti-CD23 antibodies have beenproposed that include blocking interactions other than the interactionbetween membrane CD23 and IgE. Related to this, CD23, which is a memberof the C-type lectin family, has been shown to interact with severalother ligands such as CD21, CD11b and CD11c present on a variety of celltypes including T cells and monocytes. In this context CD23 can beenvisioned as a cellular adhesion molecule.

Therefore, it has been proposed that the CD21-CD23 interaction may beinvolved in antigen presentation and subsequent IgE production. Modelssuggest CD21 on B cells sending an activation signal for IgE productionafter binding to CD23 on activated T cells present primarily in atopicindividuals. (Leconant et al., Immunol., 88:35-39 (1996); and Bonnefoyet al., Int. Amer. Allergy Immunol., 107:40-42 (1995).) Blocking thisinteraction with an anti-CD23 could block induced IgE production. (Aubryet al., Nature, 358:505-507 and Immunol., 5:944-949 (1993); Grosjean etal. (1992); Bonnefoy et al., Curr. Opin. Eur. J. Immunol., 24:2982-2988(1994); Henchoz-Lecoanet et al., Immunol., 88:35-39 (1996) Nambu et al.,Immunol. Lett., 44:163-167 (1995); Bonnefoy et al., Int. Amer. AllergyImmunol., 107:40-42 (1995).)

Yet another mechanism which would potentially explain the effects ofCD23 on IgE production involves soluble forms of CD23. It has beenreported that CD23 is cleaved from the cell surface releasing severaldifferent forms of soluble CD23 or IgE binding factors. (Sarfati et al.,Immunol., 53:197-205 (1984).) Soluble CD23 is a cytokine, with one ofits reported activities being the augmentation of IL-4 induced IgEproduction from B cells. (Pene et al., J. Cell Biochem., 39:253-269(1989); Pene et al., Eur. J. Immunol., 18:929-935 (1988); Sarfati etal., J. Immunol., 141:2195-2197 (1988); Sarfati et al. (1984) (Id.);(Saxon et al., J. Clin. Immunol. Allergy, 86 (3 pt 1) 333-344 (1990).Also, certain forms of soluble CD23 have been reported to inhibit IgEproduction (Sarfati et al., Immunol., 76:662-667 (1992)). Accordingly,anti-CD23 antibodies potentially may block IgE production by 1)inhibiting the IgE augmenting effects of soluble CD23 and/or 2) blockingthe proteolytic release of soluble CD23 from the cell surface.

Thus, based on the foregoing, it is clear that there is significantcomplexity and uncertainty in the art with respect to the functions ofmore specifically CD23 and effects on IgE production, and further withrespect to the means by which ligands specific thereto affect IgEproduction.

OBJECTS OF THE INVENTION

Thus, it is an object of the invention to produce novel ligands(antibodies) specific to CD23 and to use such antibodies to elucidatethe mechanism by which anti-CD23 antibodies modulate IgE expression.

It is another object of the invention to produce novel ligands(antibodies) which bind CD23, in particular human CD23, having improvedability to inhibit induced IgE expression.

It is a more specific object of the invention to produce anti-human CD23antibodies containing human gamma-1 constant domains.

It is another object of the invention to produce multivalent anti-humanCD23 antibodies which may be more effective by virtue of their enhancedpotential for cross linking CD23 and Fc receptors.

It is another object of the invention to provide pharmaceuticalcompositions containing anti-human CD23 monoclonal antibodies comprisinghuman gamma-1 constant domains which are capable of inhibiting inducedIgE production.

It is another object of the invention to use an anti-human CD23monoclonal antibody comprising human gamma-1 constant domains fortreatment or prophylaxsis of disease conditions wherein inhibition ofinduced IgE production is therapeutically desirable.

More specifically, it is an object of the invention to treat or preventallergic conditions, autoimmune diseases and inflammatory diseases usingan anti-human CD23 monoclonal antibody comprising human gamma-1 constantdomains.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 compares the in vitro IgE inhibitory activity of a murineanti-human CD23 monoclonal antibody (MHM6), to five primate anti-humanCD23 monoclonal antibodies (5E8, 6G5, 2C8, B3B11, and 3G12);

FIG. 2 shows that primate monoclonal antibodies 5E8 and 6G5 bind anepitope on human CD23 that is distinct from commercially availablemurine anti-human CD23 monoclonal antibody MHM6 (middle panel, FIG. 2)and compete with each other (lower panel, FIG. 2). Primate anti-humanCD23 monoclonal antibodies 2C8 and, B3B11 compete with MHM6 top panel,FIG. 2).

FIG. 3 compares the in vitro IgE inhibitory activity of a particularprimate anti-human CD23 monoclonal antibody 5E8 to four differentPRIMATIZED® versions of said primate monoclonal antibody, the sequencesof which are described below.

-   -   p5E8G4P− This PRIMATIZED® antibody contains the following        sequences: Human kappa light chain constant region and a human        gamma 4 constant region which contains a P mutation (Angal et        al., Mol. Immunol., 30:105-108 (1993));    -   p5E8G4PN− This PRIMATIZED® antibody contains the human kappa        light chain constant region and a human gamma 4 constant region        having a P mutation (Angal et al. Mol. Immunol., 30:105-108        (1993)). This antibody also contains a mutation in the heavy        chain variable region which changes an asparagine residue        (potential carbohydrate attachment site) to a lysine;    -   p5E8G1 This PRIMATIZED® antibody contains the human kappa light        chain constant region and a human gamma 1 constant region;    -   p5E8G1N− This PRIMATIZED® antibody contains the human kappa        light chain constant region and human gamma 1 constant region.        This antibody also contains a mutation in the heavy chain        variable region which changes an asparagine residue        (carbohydrate attachment site) to a lysine;

FIG. 4 contains a table which compares the apparent Kd in nM of theantibodies identified in FIG. 3 and summarizes their IgE suppressiveactivity.

FIG. 5 compares the in vitro IgE inhibitory activity of a particularprimate anti-human CD23 monoclonal antibody, 6G5, to two differentPRIMATIZED® versions of 6G5 which are described below:

-   -   p6G5G1 This PRIMATIZED® antibody contains the human lambda light        chain constant region and the human gamma 1 constant region;    -   p6G5G4P This PRIMATIZED® antibody contains the human lamda light        chain constant region and the human gamma 4 constant region with        a P mutation (Angal et al., Mol. Immunol., 30:105-108 (1993));

FIG. 6 compares the in vitro IgE inhibitory activity of primateanti-human CD23 monoclonal antibody 2C8 to F(ab′)₂ derived from 2C8;

FIG. 7 shows that the F(ab′)₂ derived from 2C8 antagonizes thesuppression of in vitro IgE activity of primate anti-human CD23monoclonal antibody 2C8.

FIG. 8 shows the in vivo IgE inhibitory activity of a particular primateanti-human CD23 monoclonal antibody, 5E8, in a SCID animal model;

FIG. 9 compares the in vivo inhibitory activity of primate anti-human6G5 and a PRIMATIZED® version thereof p6G5G4P.

FIG. 10 shows the in vivo IgE inhibitory activity of the primateanti-human CD23 monoclonal antibody 6G5 and a PRIMATIZED® versionthereof, p6G5G1.

DEFINITION OF TERMS USED IN THIS APPLICATION

Chimeric Antibody:

A recombinant antibody containing regions from two different antibodies,usually different species antibodies, most typically rodent variablesequences and human constant domain sequences.

Anti-Human CD23 Gamma 1 Antibody

An antibody that specifically binds human CD23 which contains a humangamma 1 constant region or fragment or modification thereof whichinhibits induced IgE production. This includes, in particular,antibodies containing rodent or primate variable domains or antigenbinding portions, humanized, PRIMATIZED®, and human anti-human CD23monoclonal antibodies which comprise a human gamma 1 constant domain,fragment, or modification thereof, which inhibit induced IgE productionin vitro.

PRIMATIZED® Antibody

A recombinant antibody containing primate variable sequences or antigenbinding portions, and human constant domain sequences.

Humanized Antibody:

A recombinant antibody containing a non-human variable region or antigenbinding portion which has been modified to more closely mimic a humanantibody variable region and thereby eliminate or minimize potentialimmunogenicity if administered to humans without sacrificing thespecificity or affinity of the immunoglobulin. There are several knownmethods of humanization, including, “veneering” which comprises selectmodification of surface residues, framework replacement, (CDR grafting)and molecular modeling.

Gamma 1 Constant Domain:

A particular type constant domain sequence which confers upon anantibody specific effector activities. In the present application, gamma1 constant domain refers to a human gamma 1 constant domain, fragment ormodification thereof, which retains gamma 1 effector functions incombination with anti-CD23 variable domain sequences or antigen bindingportions. Modifications include human gamma-1 constant domains whichcomprise the deletion, substitution or addition of one or more aminoacid residues. This effector function is manifested by the ability of anantibody containing such a constant domain to inhibit induced IgEproduction.

CD23:

This refers to the low affinity receptor for IgE, FceRII/CD23.

Anti-CD23 Antibody:

An antibody that specifically binds CD23, preferably human CD23.

DETAILED DESCRIPTION OF THE INVENTION

As discussed supra, while many groups have previously reported theproduction of anti-CD23 antibodies and the use thereof as antagonistsand agonists for modulating IgE production, the exact mechanism by whichsuch antibodies modulate IgE expression in systems where IL-4 inducesIgE production remains unclear. Thus, it would be beneficial if themeans by which such antibodies modulate IgE expression were elucidated,or at least better explained, as such information would be potentiallyuseful in designing therapeutics for treatment of diseases whereinmodulation of IgE production is therapeutically desirable. Inparticular, it would be beneficial if improved antibodies specific toCD23 were obtained having improved capacity to inhibit induced IgEproduction, as enhanced IgE levels are believed to be involved innumerous disease processes, e.g., allergic conditions, inflammatoryconditions and autoimmune diseases. Such diseases include by way ofexample, atopic dermatitis, eczema, allergic rhinitis and conjuntivitis,Job's syndrome, and asthma.

Toward that end, the present inventors have surprisingly discovered thatanti-human CD23 monoclonal antibodies which contain human gamma-1constant domains inhibit IgE production in systems where IgE productionis induced by IL-4 significantly better than CD23 monoclonal antibodiesof other effector types, e.g., those comprising human gamma-4 constantdomains or CD23 monoclonal antibodies or antibody fragments lackingeffector functions altogether.

While Fc mediated effector functions are sometimes significant to thetherapeutic activity of antibodies, this discovery was surprising in thecase of anti-CD23 antibodies because the role of effector function inthe IgE inhibitory activity of anti-CD23 antibodies had not beenpreviously reported. In fact, previous evidence had suggested thatantibody effector function was not significant to the ability ofanti-CD23 antibodies to inhibit induced IgE production. For example,Flores-Romo et al., Science, 261:1038-1041 (1993) had reported that Fabsprepared from a polyclonal anti-CD23 antibody inhibited an in vivoinduced IgE antigen-specific response.

This discovery was made after the present inventors isolated variousprimate antibodies specific to CD23 having anti-IgE inhibiting activityand compared these antibodies to PRIMATIZED® versions with respect totheir ability to inhibit IL-4 induced IgE production in vitro and invivo.

More specifically, and as described in greater detail infra, fiveprimate monoclonal antibodies which specifically bound both cellular andsoluble CD23 were isolated from an Old World monkey (macaque) accordingto the methodology which is disclosed in commonly assigned applicationSer. No. 08/379,072 (now allowed), which application is incorporated byreference in its entirety herein. This application described in detail ameans for producing monoclonal antibodies to desired antigens, desirablehuman antigens, in Old World monkeys and their advantages in relation toantibodies of other species as therapeutics, for example reduced orpotentially lack of immunogenicity in humans because of the phylogeneticcloseness of humans and Old World monkeys. In fact, because of thephylogenetic closeness of these species, it is difficult to distinguishOld World monkey immunoglobulins from human immunoglobulins by sequencecomparison.

Four of these five primate monoclonal anti-human CD23 antibodies weredemonstrated to be capable of inhibiting IL-4 induced IgE production inan in vitro B cell assay described in detail infra and the most potentwas also shown to inhibit IL-4 induced IgE in a SCID mouse animal model(also described in detail infra). Based on this IgE inhibitory activity,and expected low immunogenicity in humans, such antibodies arepotentially suitable as therapeutics for treating diseases whereininhibition of IgE production is therapeutically desirable.

However, in order to further reduce immunogenicity, it was elected toPRIMATIZE® two primate monoclonal antibodies (a type of chimerization ofantibodies) according to the methodology which is also described in U.S.Ser. No. 08/379,072 (now allowed), incorporated by reference herein.PRIMATIZATION® essentially refers to the production of recombinantantibodies developed by IDEC Pharmaceuticals Corporation which compriseprimate variable regions and human constant regions. Primatization ofthe two primate anti-human CD23 monoclonal (5E8 and 6G5) antibodieshaving potent IgE inhibiting activity was effected in order to eliminateany potential immunogenicity attributable to the primate constantdomains in humans.

Because of the inventors' initial expectation from published literaturethat Fc effector function was not necessary for induced IgE inhibition,human gamma 4 versions of these particular antibodies were initiallyproduced. However, quite surprisingly, it was found that the gamma-4versions produced from both of these primate monoclonal antibodies wereineffective, i.e., they required significantly higher concentrations ofPRIMATIZED® gamma 4 antibody than the primate antibody to inhibit IL-4induced IgE production in in vitro assays.

Moreover, even more surprising was the discovery that when the same twoprimate antibodies were then converted to human gamma-1 versions (bysubstitution of the primate constant domains with human gamma-1 constantdomains), that these gamma-1 antibodies very effectively inhibitedinduced IgE production in vitro. Thus, our results suggested that Fceffector function is apparently significant to the ability of anti-humanCD23 antibodies to inhibit induced IgE production. This hypothesis wasconfirmed when a third primate anti-human CD23 monoclonal, i.e., the 2C8antibody, which was shown by us to inhibit IgE production in vitro, wasconverted to a F(ab′)₂, which was found to be substantially incapable ofinhibiting induced IgE production in vitro. In fact, this F(ab′)₂ wasfound to antagonize the suppressive effects on induced IgE blockingactivity of the primate anti-human CD23 monoclonal antibody 2C8.

In addition, it was found that removing a glycosylation site in theheavy chain variable region of one of the antibodies (5E8) had no effecton binding of the antibody to CD23 (as evidenced by obtained Kd values),or on induced IgE inhibition. Thus, the differences in IgE inhibitionwere shown to apparently not involve glycosylation differences.

The PRIMATIZED® gamma 1 version of primate 6G5 was found to inhibitinduced IgE expression in SCID mice while the same concentration ofeither the primate 6G5 or the PRIMATIZED® p6G5G4p did not inhibitinduced IgE expression. Therefore, an antibody containing human gamma-1constant domains was found to be even more effective in an in vivoanimal model than the primate monoclonal antibody. Furthermore, theinventors anticipate that anti-CD23 antibodies containing human gamma-3constant domains will be just as effective as those having gamma-1constant domains, because gamma-1 and gamma-3 constant domains haveaffinity for the same classes of Fc receptors.

Accordingly, based on these results, it has been surprisingly discoveredthat an active Fc region, in particular that of human gamma 1, issignificantly involved in the mechanism of IL-4 induced IgE inhibitionby anti-human CD23 monoclonal antibodies. This discovery is quiteunexpected especially based on earlier reports that Fabs derived frompolyclonal anti-CD23 antibodies were capable of inhibiting induced IgEproduction, and also based on the various theories as to how CD23affects induced IgE expression.

Accordingly, the present invention relates to anti-human CD23 antibodiescontaining human gamma-1 constant domains and their use as therapeuticsbased on their ability to effectively inhibit IgE expression.

The skilled artisan can prepare anti-human CD23 antibodies containinghuman gamma-1 constant domains by methods which are well known in theart for the manufacture of chimeric antibodies. Essentially, suchmethods comprise producing anti-human CD23 antibodies in a desired hostor in vitro, cloning a hybridoma or cell line which produces ananti-human CD23 monoclonal antibody exhibiting desirablecharacteristics, e.g., adequate CD23 binding affinity, cloning thenucleic acid sequences which encode such antibody from said hybridoma orcell line, e.g. by polymerase chain reaction using suitable primers,isolating the variable domains contained therein, recombining suchvariable domains with human gamma-1 constant domains and the appropriatehuman light chain constant domain, and expressing the resultant nucleicacid sequence encoding a chimeric anti-human CD23 gamma-1 immunoglobulinin a suitable expression system. Preferably, the anti-human CD23antibodies of the invention will have apparent CD23 binding affinitiesranging from 0.1 nM to 1000 nM, more preferably at least 50 nM, and mostpreferably at least 5 nM.

Host cells suitable for expression of recombinant immunoglobulins arewell known in the art. For example, recombinant antibodies may beexpressed in Chinese hamster ovary (CHO) cells, DG44 or DUXB11; or CHOcells CHO K-1; mouse myeloma cells SP2/0 or X63-Ag8.653 or NSO; ratmyeloma cells YB2/0; baby hamster kidney cells, BHK; human embryonickidney line, 293; monkey kidney cells, CV1; human lung fibroblasts,WI38; human cervical carcinoma cells, HELA; insect cells, plant cells,yeast or in bacteria. Further, vectors suitable for expression ofimmunoglobulins are also well known in the art and are commerciallyavailable.

A particularly preferred vector system is the translationally impairedvector system disclosed in U.S. Ser. No. 08/147,696 (now allowed), whichcomprises a translationally impaired dominant selectable marker (neo)containing an intron into which a desired heterologous DNA is inserted.This vector system has been found to provide for very high yields ofrecombinant proteins, e.g., immunoglobulins. However, the subjectanti-CD23 antibodies may be produced in any vector system which issuitable for expression of functional immunoglobulins.

Also, the present invention embraces human monoclonal antibodies of thegamma-1 type which are specific to human CD23. Methods for isolation ofhuman monoclonal antibodies are also well known in the art and includein vitro methods, e.g., in vitro immunization of human B cells in tissueculture, and in vivo methods, e.g. synthesis of human monoclonalantibodies in SCID mice. A preferred means of producing human monoclonalantibodies in SCID mice which combines in vitro priming of human spleencells which are then introduced into SCID mice is disclosed in U.S. Ser.No. 08/488,376 (incorporated by reference in its entirety herein). Thismethod is advantageous as it provides for the reproducible recovery ofmonoclonal antibodies having high affinity against a desired antigen,e.g., a human antigen.

Also, the present invention embraces human monoclonal antibodies whichcompete with the primate anti-human CD23 monoclonal antibodies 5E8 and6G5 for binding to CD23.

EXAMPLE 1 Production of Primate Anti-CD23 Antibodies

Five primate monoclonal antibodies specific to CD23 were isolated frommacaques substantially according to the methodology disclosed in Ser.No. 08/379,072, which has been incorporated by reference herein. Theexact techniques utilized are described in detail below.

Methodology for Isolation and Characterization of Anti-Human CD23Monoclonal Antibodies

Purification of the Immunogen sCD23 from 8866 Cells

During purification, soluble CD23(sCD23) was quantified by a three-stepELISA using a murine anti-CD23 antibody (Binding Site; catalog # MC112)as a capture. The antigen was partially purified from cultures of 8866cells maintained in suspension bioreactors using RPMI 1640 (JRHBiosciences; catalog # 56-509) supplemented with 10% fetal bovine serum(JRH Biosciences) and 4 mM glutamine (JRH Biosciences; catalog # 90114)at 37° C. Carbon dioxide was used to maintain pH 7.1. After removingcells by 0.45 μm filtration, phenylmethyl sulfonyl fluoride (finalconcentration 0.2 mM, Sigman Chemical Co.; catalog # P-7626) andethylenediaminetetraacetic acid (final concentration 3 mM, SigmaChemical Co.; catalog # EDS) were added to the supernate and thesolution stored at 2-8° C. The cell-free supernate was concentratedapproximately 15 to 20-fold using a hollow-fiber ultrafiltrationcartridge (A/T Technology; catalog # UFP-10-C-9A; 10,000 d MWCO) ortangential flow ultrafiltration cartridge (Filtron Corporation; 10,000 dMWCO) at ambient temperature. The concentrated supernate was sterilefiltered and stored at -70° C. Thawed concentrates were de-lipidated byadding SM-2 BioBeads (BioRad Industries; catalog # 152-3920) at 5 g/Land stirring overnight at 2-8° C. The resin was removed by filtrationand the solution stored at 2-8° C. For some preparations of sCD23,concentrates were fractionated using ammonium sulfate (35-70% (w/v);Fisher; catalog # A702-3) before or after de-lipidation.

The de-lipidated solution was subsequently purified using affinitychromatography at 2-8° C. The affinity matrix was prepared by covalentlylinking a murine anti-CD23 monoclonal antibody (BU38) to Sepharose usingCNBr-activated Sepharose 4B (Sigma Chemical Co.; catalog # C-9142). TheBU38 antibody was purified to >90% homogeneity from ascites (BindingSite; catalog # CUS830) using Protein A chromatography. The de-lipidatedsolution was applied to the affinity column (1.5×5 cm) equilibrated with1× PBS (Gibco BRL; catalog # 70013-0.32), pH 7.2 and the column washedwith 1× PBS, pH 7.2, containing 0.05% NP40 (Sigma Chemical Co.) toremove non-bound protein. Soluble CD23 was eluted using 3.5 M MgCl₂(Fisher; catalog # M33-500). Fractions containing sCD23 were combinedand dialyzed (Baxter Spectra/Por; catalog # D1615-1) against 1× PBS, pH7.2 at 2-8° C. After dialysis, the protein solution was concentrated bycentrifugation using Centriprep 10 spin filters (Amicon Corporation;MWCO 10,000 d) and preparations stored at −70° C. The purity of sCD23was estimated to be >70% using SDS-PAGE analysis (4-20% precast gels,Novex Corporation) and Coomasie staining.

Immunization of Primates and Isolation of Immune Cells

Cynomolgus monkeys (White Sands Research Center, Alamogordo, New Mexico)were immunized with soluble CD23 which had been purified from thesupernatant of human RPMI 8866 cells (B cell lymphoma, Hassner andSaxon, J. Immunol., 132:2844 (1984)). Each monkey was immunized everythird week with 200 μg soluble CD23 in 500 μl PBS mixed with 167 μlTemuritide (adjuvant peptide) (Sigma, St. Louis, Mo., Catalog # A-9519)and 333 μl 3× PROVAX® (IDEC Pharmaceuticals Corporation). Immunizationwas effected intradermally, intraperitoneally, intramuscularly andsubcutaneously. The titer of anti-CD23 antibodies in the serum of themonkeys was measured by ELISA on 8866 cells and compared to a pre-bleedfrom the same monkeys.

Monkey PRO 978, with a serum titer of fifty thousand was sacrificed, andthe spleen and lymph nodes were surgically removed, and shipped on iceto IDEC pharmaceuticals, submerged in sterile RPMI-1640 (Gibco BRL,Gaithersburg, Md., Catalog # 21870-050) supplemented with 10% fetal calfserum, 2 mM L-glutamine, 2 mM sodium pyruvate and 50 μg/ml gentamicin.Immediately upon arrival the spleen was homogenized by squeezing itthrough a wire mesh with a glass pistil. Red blood cells were lysed inan ammonium chloride based hypotonic buffer and the remaininglymphocytes collected and washed in RPMI-1640 at least three times.Lymph nodes were homogenized similarly into a single cell suspension,collected and washed at least three times in RPMI-1640.

Production of Hybridomas

After the last wash, the cells were counted, and the primate cellsobtained above were then somatically fused to the mouse-humanheterohybridoma cell line H6K6/B5 (Carroll et al., J. Immunol. Methods,89:61 (1986)) using standard techniques (Boerner et al., J. Immunol.,147:86) (1991)) and plated into 96 well dishes (175 dishes or 14,700wells for the spleen, and 17 dishes or 1386 wells for the lymph nodes)at 300,000 cells per well.

This procedure involved the mixing of lymphocytes and theabove-identified fusion partner, at a 2:1 ratio, which cells were slowlyresuspended into 50% PEG 1500 (Sigma, Catalog # P5402) for 1 minute.These cells were then allowed to rest for 1 minute and then slowlyfurther resuspended in excess RPMI-1640. Afterward, the cells were againallowed to rest, this time for 15 minutes before a light spin at 250×g.The cells were then resuspended in RMPI-1640 growth media, which wassupplemented with 20% Fetal Calf Serum, 2 mM L-Glutamine, SodiumPyruvate, Non-Essential Amino Acids and 50 μg/ml Gentamicin, containing100 μM Hypoxanthine, 16 μM Thymidine (Boehringer Mannheim, Germany, #623091) and 5.8 μM Azaserine (Sigma, Catalog # A 1164) (HTA). HTA is aselection agent which provides the survival of successfully fused cells(primate lymphocyte fused with heterohybridoma fusion partner).

Approximately 65% of the wells showed growth (10,500 wells). These wellswere then screened for the presence of anti-human CD23 antibody by athree step cell ELISA.

ELISA Procedure

The first step of the ELISA comprised the transferral of fiftymicroliters of supernatant from each well to ninety-six well plateswhich had previously been coated with 10⁵ 8866 cells (CD23 positive cellline) per well. These plates were made by first coating the plates with50 μl of aqueous solution containing twenty pg/ml Poly L-Lysine (SigmaCatalog # P1399, MW 150,000-300,000) for thirty minutes at roomtemperature. The remaining solution was removed (“flicked out”) and theplates left to dry. Once dry, fifty μl of 8866 cells in PBS weretransferred and spun at 600 g for five minutes. The 8866 cells werecovalently bound to the plate by adding fifty μl 0.5% glutaraldehyde(Sigma Catalog # G6257) in phosphate buffered saline (PBS) for 15minutes. The glutaraldehyde was removed (flicked out) and the platesblocked with one hundred fifty μl 100 mM glycine (Sigma Catalog #G-2879) in 0.1% BSA-PBS. After the addition of supernatants, the plateswere incubated at 37° C. for one to two hours and washed seven to ninetimes with tap water, and a goat anti-human IgG antibody coupled tohorse radish peroxidase (HRPO) (Southern Biotech, Birmingham, Ala.,Catalog # 2040-05) diluted 1:2000 into 1% dry skimmed milk (Vons) inPBS-0.05% Tween 20 (Sigma, Catalog # P1379) was added. The plates wereincubated for forty-five minutes at 37° C., and again washed seven tonine times in tap water. The presence of the HRPO was detected by acolor development after the addition of a TMB reagent (Kirkegaard &Perry, Gaithersburg, Md., Catalog # 50-76-02 and 50-65-02), 100 μl/well.The reaction was stopped by adding twenty-five μl 4N H₂SO₄. Opticaldensity (OD) was measured at 470 nM on a spectrophotometer (TitertekMultiscan). The OD values greater than two times the background werescored as positive.

The second step in the ELISA was effected to confirm that thesupernatants which had been scored positive in the first ELISA reactedto CD23 and not to some irrelevant antigen. This was effected by testingthe supernatants on SupT1 cells (ERC BioServices Corporation, Rockville,Md., Catalog # 100), a CD23 negative human cell line, using the sameELISA procedure. Supernatants that scored similarly in both tests werediscarded. These results indicated that fifty-six of the 10,500 wellswith growth showed the presence of a primate monoclonal antibody thatbound to 8866 cells in two separate screenings at different times anddid not bind to SupT1 cells.

The third step of the ELISA was conducted to determine whether thesupernatants identified according to the first two ELISA steps, reactedwith soluble CD23. In this third ELISA, 96 well plates were coated at 4°C. overnight with 2 μg/ml BG-6 (Biosource International, Camarillo,Calif., Catalog # CT-CD23-CF), a mouse monoclonal antibody that binds tosoluble CD23 but does not block CD23-IgE binding, contained in a 50 mMbicarbonate buffer, pH 9.3. After removing the coating buffer, fifty μlof semi-purified soluble CD23 at a predetermined dilution in PBS wereadded to the plate and incubated for two hours at room temperature.After washing the plate with tap water seven to nine times, 50 μlsupernatants from selected wells were added. After washing the plate intap water seven to nine times, 50 μl rabbit anti-human IgG (mouseadsorbed)-HRPO (Southern Biotech, Catalog # 6145-05) diluted 1:4000 in1% dry skimmed milk in PBS with 0.05% Tween 20 were added, incubated fortwo hours at 37° C., washed seven to nine times in tap water anddeveloped with TMB as described above. Wells with OD's greater than twotimes the background were again scored as positive.

Twenty-one of the fifty-six wells that showed binding to 8866 cells alsobound to sCD23 in the ELISA. These wells were expanded and subcloned atleast twice by plating out cells at one cell per three wells. Afterapproximately three months, five stable hybridomas producing primatemonoclonal antibodies to CD23 were obtained.

Antibody Purification by Protein A Methods

Essentially, antibodies are purified by centrifugation of the culturesupernatant to remove cells and debris. The resultant centrifugedsamples are then filtered through a 0.2 μm filter. A protein A sepharoseFast flow column is then prepared and equilibrated using PBS (pH 7.4).The supernatant is then loaded on the column at an appropriate flow rate(2 ml/min). After loading, the wash column is washed with 10 columnvolume of PBS (pH 7.4). The antibody is then eluted from the column withelution buffer (0.2 M acetic acid, 0.1 M glycine pH 3.5) at 1 ml/minflow rate. One milliliter fractions/tube (2.0 M Tris-Hcl pH 10.0)including 100 μl of Tris, are then collected. Afterward,spectrophotometer readings are taken at 280 nm. The resultant fractionswith high absorbance at 280 nm containing the antibody are thencollected and dialyzed against PBS overnight. The product is thensterilized by filtration through 0.22 μm membrane and stored at −20° C.

Four of these five primate anti-human CD23 monoclonal antibodies (1H6,2C8, 5E8 and 6G5) were demonstrated to inhibit IgE production in an invitro assay which measures IgE production by IL4-hydrocortisone inducedperipheral blood mononuclear cell (PBMC) cultures. These results areshown in FIG. 1. The assay conditions are described below. The fifthprimate monoclonal anti-human CD23 antibody B3B11 was inactive in thisassay.

IL-4 Stimulated IgE Production by Peripheral Blood Mononuclear Cells

As discussed supra, the subject primate antibodies and PRIMATIZED® formsthereof were assessed for their ability to inhibit IgE production in anin vitro assay which measured the effect of such antibodies on IgEproduction by IL-4 stimulated peripheral blood mononuclear cells.

Materials for in vitro IL-4 IgE Assay

Forty-eight well flat bottom cluster plates (Costar Catalog # 3548) (1.5million PBMCs per ml per well (48 well plate))

Human recombinant IL-4 (Genzyme Catalog # 2181-01; 10 μg (2.5×10⁷units).

anti-CD23 Mabs:

-   -   murine Mab (MHM6; DAKO. Catalog # M763)    -   primate Mabs (no preservatives)    -   PRIMATIZED® (no preservatives)

HB 101 basal medium: (Irvine Scientific Catalog # T000)

HB101 supplement: (Irvine Scientific Catalog # T151)

Fetal Bovine Serum: (FBS; Bio-Whittaker Catalog # 14-501F)

dimethylsulfoxide: (DMSO; Fisher Scientific Catalog # D128-500)

hydrocortisone: (Sigma Catalog # H-0888)

puromycin: (Sigma Catalog # P-7255)

cyclohexamide: (Sigma Catalog # C-7698)

Histopaque®: (Sigma Catalog # H-8889)

Hank's Buffered Salt Solution: (HBSS; Irvine Scientific Catalog # 9232)

1% FBS in HBSS

concentrated Dulbecco's phosphate buffered saline (10× DPBS;Bio-Whittaker, Catalog # 17-517Q)

Bath Clear Microbicide (Fisher, Catalog # 13-641-334) in DPBS

Solutions:

puromycin solution: 40 μg/ml in HB101 growth medium

cyclohexamide solution: 200 μg/ml in HB101 growth medium

hydrocortisone solution: 0.1 M solution in DMSO

anti-CD23 murine Mabs were extensively dialyzed to remove preservatives

HB101 growth medium HB101 basal medium 500 ml HB101 supplement in  10 mlsterile filtered distilled H₂O  5 ml FBS  10 ml hydrocortisone solution0.25 ml  (final conc. 5 μM)

In Vitro Assay Procedure

Buffy coat cells 1:4 are diluted using HBSS at room temperature. Thesecells are derived from whole blood after an overnight incubation at roomtemperature to resolve and separate the plasma components, clottedplatelets and fibrin, and buffy coat cells.

Thirty microliters of diluted buffy coat are then overlayed onto fifteenmicroliters of Histopaque in fifty ml conical tubes. These tubes arethen centrifuged for twenty minutes at 1700 rpm at room temperaturewithout brakes (IEC 216 swinging bucket rotor). The white PBMC layer isthen collected using a sterile pipette, taking care not to disturb theother layers. The PBMCs (peripheral blood mononuclear cells) are thebuffy coat cells which have been sedimented by centrifugation partiallythrough a HISTOPAQUE® density gradient to form a distinctly visiblewhite layer of cells. These cells are collected with a pipette, rinsedwith HBSS, and then counted using a hemocytometer. Typically, 300 to 600million PBMCs can be recovered from a single 450 ml buffy coat package.

The collected PBMCs are then washed three times in 1% FBS/HBSS. Thewashed cells are collected by centrifugation for seven minutes at 1300rpm at 7° C.

The number of cells collected is then determined using a hemocytometer.The cell concentration is adjusted to about three million cells permilliliter of HB1O1 growth medium.

Approximately about 1.5 million cells (0.5 ml) are then added to eachwell of a 48 well plate. In general, five replicate samples are preparedfor each experiment. The perimeter wells of each plate are not used forcell samples. Accordingly, these wells are filled, e.g., using 0.5 ml of0.05% BathClear/DPBS.

0.5 ml HB101 growth medium containing desired amounts of IL-4 and Mab isthen added to the wells. The IL-4 used is recombinant DNA-generatedhuman interleukin 4. The Mab used in the assay is a murine, primate orPRIMATIZED® antibody. Typically, IL-4 is added at a final concentrate of100 U/ml and Mab is added at a final concentrate ranging from 0.01 to 3μg/ml.

The cells are then incubated for nine to eleven days at 37° C. in amoist incubator set at 5% CO₂. After incubation, the supernatant fluidsare collected and the IgE content is measured.

IgE ELISA

The following list identifies materials and solutions used in the IgEELISAs.

Materials and Solutions Needed for IgE ELISA

-   sulfuric acid, 4 M

coating buffer: 10 mM sodium bicarbonate buffer, pH 9.6 concentratedphosphate buffered saline (10× PBS) stock solution: NaH₂PO₄ 26.6 gmNa₂HPO₄ 289 gm NaCl 1064 gm distilled H₂O 10 L

-   blocking buffer: 10% FBS/PBS-   dilution buffer: 1% BSA/0.05% Tween 20/PBS-   washing buffer: 0.05% Tween 20/PBS-   goat anti-human IgE (epsilon chain-specific), unlabeled: (Tago    Catalog # 4104)-   human IgE standard: (The Binding Site Catalog # BP094)-   goat anti-human IgE, HRP-labeled: (Tago Catalog # AHI 0504)-   TMB peroxidase substrate: (KPL Catalog # 50-76-02)-   peroxidase solution B: (KPL Catalog # 50-65-02)-   working substrate solution: mix substrate and Solution B at 1:1    ratio-   Immulon II microtiter plates (Dynatech Labs Catalog # 011-010-3455)

IgE ELISA Procedure

Each well of a microtiter plate is coated using 100 μl of a coatingbuffer containing 2 μg/ml goat anti-human IgE.

The coated plate is then incubated overnight at 4° C.

After incubation, each well in the plate is then washed three times with200 μl of Tween 20/PBS. After washing, the non-specific binding sitesare blocked with 200 μl blocking buffer/well for 1 hour at 37° C.

One hundred μl of samples or standards are then added to each well;which wells are then incubated overnight at 4° C. After incubation, thesamples are tested with or without dilution. A standard concentrationcurve is prepared for each plate using several dilutions of IgE rangingfrom 0.1 to 50 ng/ml.

After overnight incubation, each plate is washed five times with Tween20/PBS.

One hundred μl of horseradish peroxidase (HRP) labeled goat anti-humanIgE diluted 1:10,000 in dilution buffer is then added to each drainedwell. The plate is then incubated for 4 hours at 37° C.

The plates are then washed 5 times with Tween 20/PBS and 3 times withwater.

One hundred μl of 3,3′,5,5′-tetramethylbenzidine working substratesolution is then added to each well. The plate is then incubated fortwenty-five minutes in the dark at room temperature. After incubationthe developing reaction is stopped by the addition of fifty μl of 4 Msulfuric acid.

The absorbency is then read concurrently at 450 and 540 nm. The 540 nmabsorbency values are subtracted as background.

Assay for Kd Measurement of Primate Monoclonal anti-human CD23Antibodies Scatchard Analysis Procedure

1. Radiolabeling Procedure

IODO-BEADS are washed with 100 mN Phosphate Buffer, pH 7.4 twice using 1mL of buffer per 2 beads. The beads are then dried on filter paper.

The two beads are then added to 100 μl ¹²⁵I, solution, containing about1 mCi of I, diluted with 200 μl of the phosphate buffer, and left atroom temperature for 5 minutes.

The antibody (50 μgs) is added to the preloaded beads. The reaction timefor maximal incorporation of radioactivity is 6 minutes.

The reaction is stopped by removing the radiolabeled antibody from thereaction vessel.

Gel filtration is then performed to remove excess ¹²⁵I, orunincorporated ¹²⁵I from the radiolabeled antibody solution. This iseffected by passing the radiolabeled antibody over a column made up of1.5 mL Sephadex-G25, 1.5 mL DEAE Sephadex-A25 and 0.5 mL Amberlite. Theradiolabeled antibody is eluted off in a total volume of 5 mL at aconcentration of about 10 μg/mL. (Elution Buffer: 1× PBS containing 10%Gelatin, 2% Sodium Azide and 1% BSA).

2. Optimization Assay (Direct Binding Study)

The specific activity of the 10 μg/mL radiolabeled solution isdetermined by taking a 1 μl sample and running the sample on a gammacounter.

Example

-   -   1×10⁵ cpm/μl×1000 μl/10 μg antibody        -   1×0⁵ cpm/μg antibody        -   1×0⁴ cpm/ng antibody    -   Molecular wt. of antibody=75,000 ng/nmole    -   Specific activity:        -   1×0⁴ cpm/ng×75,000 ng/nmole=7.5×0⁸ cpm/nmole

The antigen-coated plate is blocked (to eliminate non-specific binding,e.g., with mB7.1-CHO) and the background plate (i.e., Untransfected-CHO)for one hour at room temperature-with 200 μl/well of blocking buffer(Blocking Buffer: 1× PBS containing 10% Gelatin, 2% Sodium Azide, 1% BSAand 10% FBS).

The plate(s) are then washed, typically ten times by hand with tapwater.

The 10 μg/mL radiolabeled antibody (50 μls) is then titrated by two-foldserial dilutions across the plate(s) using a multichannel pipette.Incubate for one hour at room temperature.

The plate(s) are again washed about 6-7 times with 200 μl/well of washbuffer (Wash Buffer:—1× PBS containing 10% Gelatin and 2% Sodium Azide).

The radioactivity counts in each well are then determined by running thewells on a gamma counter.

The optimal radiolabeled antibody concentration is the concentration inwhich the difference between the specific counts and background countsis at a maximum.

3. Scatchard Analysis of Competition Assay

The 10 μg/mL radiolabeled solution is diluted to the optimalconcentration determined in the Direct Binding experiment.

The antigen-coated plate and the background plate are blocked for onehour at room temperature with 200 μl/well of blocking buffer.

The plate(s) are then washed, e.g., about 10 times, by hand with tapwater.

The “cold” (no radiolabel) antibody is then titrated by two-folddilutions in a separate U-bottom microtitre plate. The startingconcentration of the “cold” antibody should be at least 100 timesgreater than that of the optimal radiolabeled antibody concentration.

Example

-   -   Optimal Radiolabeled Conc.: 0.5 μg/mL    -   “Cold” Antibody Conc.: 100 μg/mL (Note: 1:2 titration in the        first well will adjust the “cold” antibody concentration to 50        μg/mL.)

Fifty μl/well of optimal radiolabeled antibody are then added to thewells containing “cold” antibody.

One hundred μl/well of the mixed solution are then transferred to thecorresponding wells of the antigen-coated plate, and incubated for onehour at room temperature.

Also, it is desirable also that the following controls be effected:

a) Direct binding of radiolabeled antibody to antigen-coated plate (5wells),

b) Direct binding of radiolabeled antibody to background plate (5wells).

After incubation, the plate(s) are washed, e.g., about 6-7 times, with200 μl/well of wash buffer.

The radioactivity counts in each well are then determined by running thewells on a gamma counter.

These calculations are determined by calculating the specific counts ineach well tested by subtracting the background counts from the countsbound to the antigen-coated plate.

4. Calculations for Scatchard Analysis

The Molar Concentration of Bound antibody [B] can then be determined asfollows:

Example: At 50 μg/mL “cold antibody”

-   Specific counts bound: 4382 cpm-   Counts bound in the presence of 50 μg/mL “cold” ab: 215 cpm-   Difference: 4382 cpm−215 cpm=4167 cpm-   Specific Activity (radiolabeled ab): 5.54×10⁹ cpm/nmole-   4167 cpm÷5.54×10⁹ cpm/nmole=7.52×10⁻⁷ nmole-   7.53×10⁻⁷ nmole÷0.05 mL (sample vol.) =1.50×10⁻⁵ nmole/mL =1.50×10⁻⁸    pmole/mL [B]=1.50×10⁻¹¹ mole/mL (M)    Total Molar Concentration [T] is determined as follows:    $\begin{matrix}    \begin{matrix}    {{50\quad\mu\quad{g/{mL}} \times 1\quad\mu\quad{{mole}/75}},{{000\quad\mu\quad g} = {6.67 \times 10^{- 4}\quad\mu\quad{{mole}/{mL}}}}} \\    {= {6.67 \times 10^{- 7}{{mmole}/{mL}}\quad(M)}} \\    {= {66667 \times 10^{- 11}{{mmole}/{mL}}\quad(M)}}    \end{matrix} & \lbrack T\rbrack    \end{matrix}$    Free antibody [F] is determined as follows: $\begin{matrix}    {{{{Free}\quad{Molar}\quad{{Conc}.}} = {{Total}\quad{minus}\quad{Bound}}}\text{}\begin{matrix}    {\quad{= {\left( {66667 \times 10^{- 11}} \right) - \left( {1.50 \times 10^{- 11}} \right)}}} \\    {= {66665.5 \times 10^{- 11}{{mmole}/{mL}}\quad(M)}} \\    \quad    \end{matrix}} & \lbrack F\rbrack    \end{matrix}$    Calculate B/F.    Plot B versus B/F on Cricket Graph software.

Activity and Affinity of Anti-Human CD23 Antibodies According to theInvention

Four of the five isolated primate anti-human CD23 monoclonal antibodies(B3B11, 2C8, 5E8 and 6G5) were found to inhibit IgE production in theabove-identified in vitro assay which measures IgE production byIL4-hydrocortisone induced peripheral blood mononuclear cell (PBMC)cultures. These results are shown in FIG. 1. The fifth primatemonoclonal anti-human CD23 antibody 3G12 was inactive in this assay.

Two of the four primate monoclonal anti-human CD23 antibodies (B3B11 and2C8) found to be active in this in vitro assay were found to competewith a commercially available mouse anti-human CD23 antibody MHM6 (CAKOA/S, Glostrup, Denmark Catalog # M763). (FIG. 2, top panel.) However, inrepeated assays these antibodies were not as potent IgE inhibitors asMHM6 (data not shown). By contrast, the other primate anti-CD23monoclonal antibodies (5E8 and 6G5) were found to compete with eachother and did not complete with MHM6. (FIG. 2, middle and bottompanels.) Moreover, the primate anti-human CD23 monoclonal antibody 5E8was found to be a potent inhibitor of IL-4 induced IgE in the in vitroassay. (See FIGS. 1 and 3)

Modified Hu-SCID-Mouse Model for Human IgE Synthesis and Measuring theInhibition of IL-4 Induced IgE Production by Anti-CD23 Antibodies InVivo

A modified hu-PBMC-SCID mouse model was also developed to detect theeffect of the subject antibodies on induced human IgE production invivo. PBMCs obtained from two donors were cultured with IL-4 in vitrofor two days. PBMCs were pooled and used to reconstitute groups ofC.B.-17 SCID mice with and without antibodies. Mice were bled on day 14,21, 28 and 35 and serum IgG and IgE levels were determined by ELISA.This in vivo model was used to assay primate and two different versionsof PRIMATIZED® antibodies to CD23 for their ability to inhibit theproduction of IgE.

A modified SCID mouse model was used because it is known that severecombined immunodeficiency scid/scid (SCID) mice, C.B.-17 (Bosma et al.,Nature, 301:527 (1983)) reconstituted with human peripheral bloodmononuclear cells (hu-PBMC-SCID) can produce significant quantities ofhuman immunoglobulins (Ig) (Moiser et al., Nature, 335:256 (1988);Moiser et al., J. Clin. Immunol., 10:185 (1990); Abedi et al., J.Immunol., 22:823 (1992); and Mazingue et al., Eur. J. Immunol., 21:1763(1991).) The predominant isotype of human immunoglobulin (Ig) producedin hu-PBMC-SCID mice is IgG. Generally, IgM, IgA and IgE isotypes arefound in very low or non-detectable levels except in cases where PBMC isobtained from donors with certain autoimmune or allergic diseaseconditions. It has also been reported that manipulation of hu-PBMC SCIDmouse model with certain cytokines may be provided for the generation ofsignificant levels of non-IgG isotypes, including IgE (Kilchherr et al.,Cellular Immunology, 151:241 (1993); Spiegelberg et al., J. Clin.Investigation, 93:711 (1994); and Carballido et al., J. Immunol.,155:4162 (1995)). The hu-PBMC-SCIDs, has been also used to generateantigen specific Ig provided the donor has been primed for the antigenin vivo.

Therefore, the aim of the present inventors was focused on establishinga suitable human IgE producing hu-PBMC-SCID mouse model that could beused to test the efficacy of therapeutic for treatment of IgE relateddiseases such as allergic disorders, including the subject anti-CD23antibodies.

Materials and Methods:

The following materials and methods were used in the hu-PBMC-SCID mousemodel described below.

SCID mice: C.B-17 scid/scid immunodeficient mice were obtained fromTaconic (C.B.-17/IcrTac-scidfDF) and maintained in IDEC Pharmaceuticals'animal facility. Mice were housed in sterilized microbarrier units withsterilized bedding. Animal studies were performed in accordance with the“Guide for the Care and Use of Laboratory Animals” specified by theCommittee on Care of Laboratory Animal Resources Commission on LifeScience-National Research Council (Guide for the Care and Use ofLaboratory Animals, DHHS Publ. No. (NIH) 86-23, Bethesda, Md., NIH,1985).

Human PBMC: PBMCs were isolated from buffy coats obtained from a bloodbank by centrifugation through Ficoll-Hypaque (Histopaque-1077) asrecommended by the manufacturer (Sigma Diagnostics Catalog # 1077-1).Lymphocyte preparation at the interface of the gradient were harvestedand washed three times in Hanks Balanced Salt Solution (HBSS)(Bio-Whittaker Catalog # 10-527F). For each experiment PBMCs wereobtained from two separate donors and cultured separately in vitro.PBMCs were resuspended at 1-3×10⁶ cells/ml concentration in HB-Basalmedium plus 1% HB101 lyophilized supplement (Irvine Scientific Catalog #T000 & T151) containing 5% FCS plus 1000 IU/ml of IL-4 (Genzyme, Inc.Catalog # 2181-01) and incubated for 48 hours at 37° C. with 5% CO₂.After incubation, the cells from different buffy coats were harvested,pooled and used to reconstitute SCID mice.

In Vivo Assay Conditions

Groups of mice (four to five per group) were injected withfifty-sixty×10⁶ lymphocytes in 200-300 μl volume of HBSSintraperitoneally (i.p.) on day zero. For the groups that receivedanti-CD23 antibody, on day zero, PBMCs were mixed with anti-CD23antibody (200 to 400 μg/mouse) before i.p., injection and the secondinjection was given on day seven. All mice received 5000 IU per mouse ofIL-4 i.p., between day zero to day five. A group which was not injectedwith antibody served as the control group. Mice were bled from aretro-orbital vein and the serum was analyzed for IgG and IgE on daysfourteen, twenty-one, twenty-eight and thirty-five by ELISA.

FIG. 8 shows that the primate anti-human CD23 monoclonal antibody 5E8 iseffective in inhibiting IL-4 induced IgE production in vivo in the SCIDmouse model.

Cloning and Expression of PRIMATIZED®(anti-human CD23 MonoclonalAntibodies

In order to clone primate immunoglobulin variable domains, Poly A+ RNAwas separately isolated from approximately 2×10⁶ cells from the primateheterohybridomas secreting the anti-human CD23 monoclonal antibodies 6G5and 5E8 by using the Micro-FastTrack mRNA isolation Kit (InvitrogenCatalog # K1520-02) according to methods set forth by the manufacturer.

The first strand of cDNA was synthesized from the poly A+ RNA by usingthe cDNA Cycle Kit (Invitrogen Catalog # L1310-01) according toconventional methods.

The light and heavy chain variable regions of 6G5 and 5E8 were thenisolated by PCR from cDNA using PCR primers that were selected basedupon different consensus families of human immunoglobulins. 5′ primerswere selected which corresponded to the beginning of the leadersequences of the light and heavy variable region and 3′ primers wereselected which corresponded to the J region (The specific primers usedto PCR amplify the lambda light chain variable domain of 6G5, the kappalight chain variable domains of 5E8, and the heavy chain variabledomains of 6G5 and 5E8 are set forth in Tables 1-3). PCR was performedaccording to standard methods (30 cycles with 1 minute at 94° C., 1.5minutes at 54° C. and 2 minutes at 72° C. in a Hot start 100 tube (GibcoBRL Catalog # 10332-013). PCR was set up in 50 μl reactions containing 5μl out of 80 μl cDNA (from 2×10⁶ cells) as a template, 2 μl of 5 nMdNTP, 1 μl of Taq polymerase, 5 μl of Taq polymerase buffer, 2 μl of the5′ primer (25 pmoles/μl), 2 μl of the 3′ primer (25 pmoles/μl), and 36μl of water. (Taq polymerase and buffer were obtained from StratageneCatalog # 600131, dNTP from Boehringer Mannheim Catalog #1581295.)

A) Construction of the plasmids N5LG1+6G5 and N5LG4P+6G5

1) Cloning the Light Chain Variable Domain of Primate Monoclonalanti-human CD23 Antibody 6G5 by PCR

The first PCR amplification of the light chain variable region from thecDNA of primate monoclonal antibody 6G5 showed bands which wereconsistent in situ with the lambda light chain variable region. Thesebands appeared in all reactions using the three different early leadersequence primers. (See Tables 1-3.) However, the PCR product obtainedusing primer 745 (Family 2) was considered more specific because of therelatively greater intensity of the PCR product band.

This PCR product was isolated using a Qiaquick Gel Extraction Kit(Qiagen Catalog # 28704). The purified PCR fragment was digested withBgl II and Avr II restriction endonucleases, and ligated into themammalian expression vector N5LG1 which was digested with the samerestriction endonucleases. Twenty microliters of the ligation mixturecontaining the purified PCR product from one fifty microliter PCRreaction, 100 mg N5LG1 vector, two microliters of 10× ligation buffer(NEB Catalog # 202S) and two microliters of T4 ligase (NEB Catalog #202S), were then incubated at 14° C. overnight.

The mammalian expression vector N5LG1 contains genetic sequences (e.g.,regulatory sequences, coding sequences) which provide for the expressionof four separate proteins in a mammalian cell. They are:

(i) a partial immunoglobulin light chain with the human lambda lightchain constant region and unique restriction endonuclease sites forinserting light chain variable domains;

(ii) a partial immunoglobulin heavy chain with the and human gamma 1chain constant region coding sequences and unique restrictionendonuclease sites for inserting heavy chain variable domains;

(iii) a neomycin phosphotransferase gene used to select for cells thathave incorporated the plasmid and are resistant to the antibioticGeneticin (Gibco BRL Catalog # 10131-1209); and

(iv) a murine dihydrofolate reductase gene (DHFR) which provides for theselection and genomic amplification when cells are cultured in thepresence of methotrexate (MTX, Sigma Catalog # A-6770) (Reff et al.,Blood, 83:433-445 (1994).

After ligation, the mixture was digested using Pme I restrictionendonuclease, which digests the parent N5LG1 plasmid, but not the N5LG1plasmid which has been ligated to the light variable domain of 6G5.After digestion, the mixture was transformed into Epicurian coli®XL1-Blue competent cells (Stratagene Catalog # 200249) as follows.

One hundred microliters of competent cells were mixed with 10 μl of theabove ligation mixture, set on ice for 30 minutes, then heated at 45° C.for 30 seconds. This mixture was placed on ice for 2 minutes, and 900 μlof SOC, prewarmed to room temperature, was then added. (SOC is LB brothGibco BRL Catalog # 10855-013, plus 0.02 M MgCl₂, 0.02 M MgSO₄ and 0.02M D-glucose.) After incubation at 37° C. for an hour, the mixture wascentrifuged at 4000 g for a minute, and 800 μl of supernatant discarded.The rest of the mixture was plated onto a LB agar (Gibco BRL Catalog #12945-044) dish containing 50 μg/ml ampicillin (Amp, Gibco BRL Catalog #13075-015). Plasmid DNA was isolated from individual colonies of E. colithat grew on the Amp plate by using the Wizard® Miniprep DNApurification system (Promega Catalog # A7510).

The isolated plasmid DNA was then characterized by digestion with Bgl IIand Avr II followed by agarose gel electrophoresis. An ethidium stainedDNA band of 400 bp was indicative of a potential successful cloning of alight chain variable domain.

To confirm this was an immunoglobulin light chain variable domain,sequencing was done using the Sequenase 7-Deaza-dGTP DNA Sequencing Kit(USB catalog # 70990) with sequencing primers 607 and GE 108. (SeeSequencing Primers in Table 4.)

A second independent PCR amplification of the light chain from cDNA ofprimate monoclonal antibody 6G5 was effected using a 5′ primer earlyleader sequence of lambda light chain family 2 (primer 745) (SEQ ID NO:15) and the 3′ J region primer 926 (SEQ ID NO: 17). (See Primers for PCRof the lambda light chain variable domain of 6G5 in Tables 1-3 (SEQ IDNOs: 9-25). The isolated PCR product (see technique above) was clonedinto TA vector by using the Original TA Cloning( Kit (Invitrogen Catalog# K2000-01). The isolated miniprep DNA (see technique above) wasexamined under agarose gel electrophoresis after digestion with EcoR Irestriction endonuclease. The resultant PCR product comprised in the TAvector was then sequenced (as described previously) using Sp6 (SEQ IDNO: 26) and M13(-40) (SEQ ID NO: 27) forward primers (See Sequencingprimers in Table 4 (SEQ ID NOs: 26-35)). The resultant light chainsequence was identical to that of light chain from the first PCR. Thisentire sequence of the light chain variable domain of primate monoclonalanti-human CD23 antibody 6G5 is presented below (SEQ ID NOs: 1-2). Lightchain variable region of primate monoclonal antibody anti-human CD23 6G5Leader Met Ala Trp Thr Leu Leu Leu Val Thr Leu Leu Thr Gln Gly Thr ATGGCC TGG ACT CTG CTC CTC GTC ACC CTC CTC ACT CAG GGC ACA              −1Gly Ser Trp Ala (SEQ ID NO: 2 - residues 1-19) GGA TCC TGG GCT (SEQ IDNO: 1 - bases 1-57)

Mature Protein (Numbering is Kabat) Framework 1  1                              9  11 Gln Ser Ala Pro Thr Gln Pro ProSer Val Ser Gly Ser Pro Gly CAG TCT GCC CCG ACT CAG CCT CCC TCT GTG TCTGGG TCT CCT GGA              20          23 Gln Ser Val Thr Ile Ser Cys(SEQ ID NO: 2 - residues 20-41) CAG TCG GTC ACC ATC TCC TGC (SEQ ID NO:1 - bases 58-123) CDR 1  24           27 27A 27B 27C   34 Thr Gly ThrSer Asp Asp Val Gly (SEQ ID NO: 2 -  28 Gly Tyr Asn Tyr Val Ser residues42-55) ACT GGA ACC AGC GAT GAC GTT GGT (SEQ ID NO: 1 - GGT TAT AAC TATGTC TCC bases 124-165) Framework 2  35                  40 Trp Tyr GlnHis His Pro Gly Lys (SEQ ID NO: 2 -                          49 Ala ProLys Leu Met Ile Tyr residues 56-70) TGG TAC CAA CAC CAC CCA GGC AAA (SEQID NO: 1 - GCC CCC AAA CTC ATG ATT TAT bases 166-210) CDR2 50                      56 Asp Val Ala Lys Arg Ala Ser (SEQ ID NO: 2 -residues 71-77) GAT GTC GCT AAG CGG GCC TCA (SEQ ID NO: 1 - bases211-231) Framework 3  57          60 Gly Val Ser Asp Arg Phe Ser Gly SerLys Ser Gly      70 Asn Thr Ala GGG GTC TCT GAT CGC TTC TCT GGC TCC AAGTCT GGC AAC ACG GCC Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu AlaAsp Tyr TCC CTG ACC ATC TCT GGG CTC CAG GCT GAG GAC GAG GCT GAT TAT     88 Tyr Cys (SEQ ID NO: 2 - residues 78-109) TAC TGT (SEQ ID NO: 1 -bases 232-327) CDR 3  89  90                  95 95A Cys Ser Tyr Thr ThrSer Ser Thr (SEQ ID NO: 2 -  96  97 Leu Leu residues 110-119) TGT TCATAT ACA ACC AGT AGC ACT (SEQ ID NO: 1 - TTG TTA bases 328-357) Framework4  98     100  107 Phe Gly Arg Gly Thr Arg Leu Thr (SEQ ID NO: 2 -    106 106A Val Leu Gly residues 120-130) TTC GGA AGA GGG ACC CGG TTGACC (SEQ ID NO: 1 - GTC CTA GGT bases 358-390)

2) Cloning the Heavy Chain Variable Domain of Primate Monoclonalanti-human CD23 Antibody 6G5 by PCR

The first PCR amplification of the heavy chain variable domain from cDNAof primate monoclonal antibody 6G5 was performer by using the set ofearly leader sequence primers described supra and the 3′ J region primerGE244 (SEQ ID NO: 23). These primers are in Tables 1-3 (SEQ ID NOs:9-25) infra. This reaction resulted in a 350 base PCR product. This 350base product (purified as described supra), was digested with Nhe I andSal I, and ligated into N5LG1 and digested with the same endonucleasesin the first PCR amplification. The resultant ligation mixture wastransformed into host cells using the same techniques for cloning thelight chain. Plasmid N5LG1 containing the 350 base PCR product was thenisolated and sequenced (using sequencing primers 266 (SEQ ID NO: 32) and268) (SEQ ID NO: 33). (These Sequencing primers are set forth in Table 4(SEQ ID NOs: 26-35).) Sequencing revealed that the PCR product containedonly part of the heavy variable domain and comprised a deletion in itsamino terminus (Sequence began at framework 2, codon 36).

A second independent PCR reaction was conducted to amplify and isolatethe heavy chain variable domain of primate monoclonal antibody 6G5 usinga 5′ early leader sequence primer for family 1 (MB1503) (SEQ ID NO: 18)and a 3′ J′ region primer GE244 (SEQ ID NO: 23). (These primers are alsocontained in Tables 1-3 (SEQ ID NOs: 9-25)) The resultant PCR productwas then cloned into the NSLG1 using the same techniques describedsupra. Its sequence was found to be identical to the first PCR product.

Therefore, in order to clone the whole heavy variable domain of 6G5including the missing 5′ terminus a new longer 3′ primer (MB1533) (SEQID NO: 25) which included the CDR3 and framework 4 regions of the 6G5heavy variable chain was then used in a third independent PCR reactionwith the family 1 5′ primer (MB1503) (SEQ ID NO: 18). (These primers arealso contained in Tables 1-3 (SEQ ID NOs: 9-25).) After PCR, a larger420 base PCR product was observed on the agarose gel. This PCR productwas isolated as described previously, and cloned into a TA vector. Theresultant PCR product contained in the TA vector was then sequenced.Sequencing revealed that this DNA contained the whole heavy variabledomain and that the 3′ part was identical to that of previously clonedpartial heavy chain variable domain from the first two PCR reactions.

A fourth independent PCR was performed using the same primers as thethird PCR amplification. This resulted in a PCR product which wasisolated and cloned into the TA vector as described previously. Thesequence of the fourth independent PCR product was found to be identicalto that obtained in the third PCR amplification. This sequence, whichcomprises the heavy chain variable domain of primate monoclonalanti-human CD23 antibody 6G5, is presented below (SEQ ID NOs: 3-4).Heavy chain variable region of primate monoclonal antibody anti-humanCD23 6G5 Leader Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala ProArg ATG AAA CAC CTG TGG TTC TTC CTC CTC CTG GTG GCA GCT CCC AGA             −1 Trp Val Leu Ser (SEQ ID NO: 4 - residues 1-19) TGG GTCCTG TCC (SEQ ID NO: 3 - bases 1-57)

Mature Protein (Numbering is Kabat) Framework 1  1                                  10 Gln Leu Gln Leu Gln Glu Ser GlyPro Gly Val Val Lys Pro Ser CAG CTG CAG CTG CAG GAG TCG GGC CCA GGA GTGGTG AAG CCT TCG              20 Glu Thr Leu Ser Leu Thr Cys Ala (SEQ IDNO: 4 -                          30 Val Ser Gly Gly Ser Val Ser residues20-49) GAG ACC CTG TCC CTC ACC TGC GCT (SEQ ID NO: 3 - GTC TCT GGT GGCTCT GTC AGC bases 58-147) CDR 1  31              35 35a Ser Ser Asn TrpTrp Thr (SEQ ID NO: 4 - residues 50-55) AGT AGT AAC TGG TGG ACC (SEQ IDNO: 3 - bases 148-165) Framework 2  36              40 Trp Ile Arg GlnPro Pro Gly Lys (SEQ ID NO: 4 -                      49 Gly Leu Glu TrpIle Gly residues 56-69) TGG ATC CGC CAG CCC CCA GGG AAG (SEQ ID NO: 3 -GGA CTG GAG TGG ATT GGA bases 166-207) CDR2  50      5252A  53                          60 Arg Ile Ser Gly Ser Gly Gly Ala ThrAsn Tyr Asn Pro Ser Leu CGT ATC TCT GGT AGT GGT GGG GCC ACC AAC TAC AACCCG TCC CTC  65 Lys Ser (SEQ ID NO: 4 - residues 70-86) AAG AGT (SEQ IDNO: 3 - bases 208-258) Framework 3  66              70 Arg Val Ile IleSer Gln Asp Thr Ser Lys Asn Gln          80 Phe Ser Leu CGA GTC ATC ATTTCA CAA GAC ACG TCC AAG AAC CAG TTC TCC CTG      82 82a 82b 82c  83 AsnLeu Asn Ser Val Thr Ala Ala Asp Thr Ala Val  90 Tyr Tyr Cys AAC CTG AACTCT GTG ACC GCC GCG GAC ACG GCC GTG TAT TAC TGT      94 Ala Arg (SEQ IDNO: 4 - residues 87-118) GCC AGA (SEQ ID NO: 3 - bases 259-354) CDR 3 95                  100 100a 102 Asp Trp Ala Gln Ile Ala Gly Thr (SEQID NO: 4 - 100b 100c 100d 101 Thr Leu Gly Phe residues 119-130) GAT TGGGCC CAA ATA GCT GGA ACA (SEQ ID NO: 3 - ACG CTA GGC TTC bases 355-390)Framework 4 103 113 Trp Gly Gln Gly Val Leu Val Thr (SEQ ID NO: 4 - 110Val Ser Ser residues 131-141) TGG GGC CAG GGA GTC CTG GTC ACC (SEQ IDNO: 3 - GTC TCC TCA bases 391-423)3) Construction of Mammalian Expression Vectors

In order to insert the cloned heavy chain variable domains of 6G5 into amammalian expression vector, the heavy chain variable domain in the TAvector (obtained in the 3rd independent PCR) was digested with Nhe I andSal I and cloned into the N5LG1 vector which was digested with the samerestriction enzymes and which vector already contains the light chainvariable domain. The resultant mammalian expression vector was namedN5LG1+6G5.

To construct the N5LG4P+6G5 vector, both the light and heavy chainvariable domains were isolated from N5LG1+6G5 by digestion of Bgl II andAvr II, and Nhe I and Sal I respectively. The mammalian expressionvector N5LG4P vector is identical to the N5LG1 vector described above,except the human gamma 1 was replaced with a human gamma 4 constantregion containing a mutation of a serine to a proline in the hingeregion to increase stability of the immunoglobulin and improvepharmacokinetics in vivo (“P” mutation). The light chain variable domainwas cloned in the plasmid first and the heavy chain variable domain wascloned into the vector containing the light chain variable domain usingtechniques previously described. This mammalian expression vector wasnamed N5LG4P+6G5.

B. Construction of the plasmids N5KG4P+5E8, N5KG1+5E8, N5KG4P+5E8N−, andN5KG1+5E8N−

1. Cloning the Light Chain Variable Domain of Primate Monoclonalanti-human CD23 Antibody 5E8 by PCR

The first PCR reaction of the light chain variable domain from FEE cDNAwas carried out using a set of kappa early leader sequence primers andthe 3′ J region primer GE204 (SEQ ID NO: 13). (See primers for PCR ofthe kappa light chain variable domain of 5E8 in Tables 1-3 (SEQ ID NOs:9-25)). A 420 base PCR product was obtained. The isolated 420 base PCRproduct was digested with Bgl II and BsiW I restriction endonucleases,cloned into the mammalian expression vector N5KG4P and sequenced usingGE108 (SEQ ID NO: 29) and 377 (SEQ ID NO: 30) primers (which arecontained in Table 4 (SEQ ID NOs: 26-35)): The mammalian expressionvector N5KG4P is identical to the vector N5LG4P except it contains thehuman kappa light chain constant region in place of the human lambdalight chain constant region. Sequencing of this 420 polynucleotide DNArevealed that it contains the entire kappa light chain variable domain.

A second independent PCR of the light chain variable region wasperformed using the 5′ family 1 primer GE201 (SEQ ID NO: 9) and the 3′primer GE204 (SEQ ID NO: 13). (See primers for PCR of the kappa lightchain variable domain of 5E8 in Tables 1-3 (SEQ ID NOs: 9-25)). Theisolated PCR product was cloned into the TA vector (using methodspreviously described) and sequenced using Sp6 (SEQ ID NO: 26) and T7promoter (SEQ ID NOs: 28) primers. Sequencing revealed that this PCRproduct was identical to that obtained from the first PCR. The entiresequence of the light chain variable domain of primate monoclonalanti-human CD23 antibody 5E8 is presented below (SEQ ID NOs: 5-6). Lightchain variable region of primate monoclonal antibody anti-human CD23 5E8Leader Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu ATGGAC ATG AGG GTC CCC GCT CAG CTC CTG GGG CTC CTT CTG CTC                         −1 Trp Leu Pro Gly Ala Arg Cys (SEQ ID NO: 6 -residues 1-22) TGG CTC CCA GGT GCC AGA TGT (SEQ ID NO: 5 - bases 1-66)

Mature Protein (Numbering is Kabat) Framework 1  1                                  10 Asp Ile Gln Met Thr Gln Ser ProSer Ser Leu Ser Ala Ser Val GAC ATC CAG ATG ACC CAG TCT CCA TCT TCC CTGTCT GCA TCT GTA                  20          23 Gly Asp Arg Val Thr IleThr Cys (SEQ ID NO: 6 - residues 23-45) GGG GAC AGA GTC ACC ATC ACT TGC(SEQ ID NO: 5 - bases 67-135) CDR 1  24                         30 34Arg Ala Ser Gln Asp Ile Arg Tyr (SEQ ID NO: 6 - Tyr Leu Asn residues46-56 amino acids 24-34) AGG GCA AGT CAG GAC ATT AGG TAT (SEQ ID NO: 5 -TAT TTA AAT bases 136-168) Framework 2  35                  40 Try TyrGln Gln Lys Pro Gly Lys (SEQ ID NO: 6 -                          49 AlaPro Lys Leu Leu Ile Tyr residues 57-71) TGG TAT CAG CAG AAA CCA GGA AAA(SEQ ID NO: 5 - GCT CCT AAG CTC CTG ATC TAT bases 169-213) CDR2 50                      56 Val Ala Ser Ser Leu Gln Ser (SEQ ID NO: 6 -residues 72-78) GTT GCA TCC AGT TTG CAA AGT (SEQ ID NO: 5 - bases214-234) Framework 3  57          60 Gly Val Pro Ser Arg Phe Ser Gly SerGly Ser Gly      70 Thr Glu Phe GGG GTC CCA TCA AGG TTC AGC GGC AGT GGATCT GGG ACA GAG TTC                                  80 Thr Leu Thr ValSer Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr ACT CTC ACC GTC AGC AGC CTGCAG CCT GAA GAT TTT GCG ACT TAT      88 Tyr Cys (SEQ ID NO: 6 - residues79-110) TAC TGT (SEQ ID NO: 5 - bases 235-330) CDR 3  89  90 Leu Gln ValTyr Ser Thr Pro Arg (SEQ ID NO: 6 -  97 Thr residues 111-119) CTA CAGGTT TAT AGT ACC CCT CGG (SEQ ID NO: 5 - ACG bases 331-357) Framework 4 98     100 Phe Gly Gln Gly Thr Lys Val Glu (SEQ ID NO: 6 -     107 IleLys residues 120-129) TTC GGC CAA GGG ACC AAG GTG GAA (SEQ ID NO: 5 -ATC AAA bases 358-387)2) Cloning the Heavy Chain Variable Domain of Primate Monoclonalanti-human CD23 Antibody 5E8 by PCR

The first PCR of the heavy chain variable domain of 5E8 was performedusing a set of 5′ early leader heavy chain sequence primers and the 3′primer GE210 (SEQ ID NO: 24). (See primers for PCR of the heavy chainvariable domain of 6G5 and 5E8 in Table 1 (SEQ ID NOs: 9-13)). A 420base PCR product appeared in the family 3 primer reaction. The PCRproduct was purified and then digested with Nhe I and Sal I and clonedinto the mammalian expression vector N5KG4P vector (as describedpreviously). The PCR product was sequenced using the 268 (SEQ ID NO: 33)and 928 (SEQ ID NO: 35) primers. (See sequencing primers in Table 4 (SEQID NOs: 26-35).)

A second independent PCR of the heavy chain variable domain of 5E8 wasperformed using the family 3 5′ primer GE207 (SEQ ID NO: 20) and the 3′primer GE210 (SEQ ID NO: 24). (See primers for PCR of the, heavy chainvariable domain of 6G5 and 5E8 in Tables 1-3 (SEQ ID NOs: 9-25)). Theisolated PCR product was cloned into a TA vector using the sametechniques previously described and sequenced by-using Sp6 (SEQ ID NO:26) and T7 (SEQ ID NO: 28) primers. Sequencing revealed that the TAC atcodon 91 had been changed into TGC.

In order to determine the appropriate codon at 91, a third independentPCR was performed using the same primers as the second PCR (see above).The PCR product was again cloned into a TA vector and sequenced usingSp6 (SEQ ID NO: 26) and T7 (SEQ ID NO: 28) primers. The sequence wasfound to be identical to the heavy chain variable sequence obtained inthe first PCR. Therefore, the TGC at position 91 in the secondindependent PCR product is apparently the result of an error introducedduring PCR. This entire sequence of the heavy chain variable domain ofprimate monoclonal anti-human CD23 antibody 6G5 is presented below (SEQID NOs: 7-8). Heavy chain variable region of primate monoclonal antibodyanti-human CD23 5E8 Leader Met Glu Phe Gly Leu Ser Trp Val Phe Leu ValPro Leu Leu Lys ATG GAG TTT GGG CTG AGC TGG GTT TTC CTT GTT CCT CTT TTGAAA              −1 Gly Val Gln Cys (SEQ ID NO: 8 - residues 1-19) GGTGTC CAG TGT (SEQ ID NO: 7 - bases 1-57)

Mature Protein (Numbering is Kabat) Framework 1  1                                  10 Glu Val Gln Leu Val Glu Ser GlyGly Gly Leu Ala Lys Pro Gly GAG GTG CAG CTG GTG GAG TCT GGG GGC GGC TTGGCA AAG CCT GGG                  20 Gly Ser Leu Arg Leu Ser Cys Ala (SEQID NO: 8 -                          30 Ala Ser Gly Phe Arg Phe Thrresidues 20-49) GGG TCC CTG AGA CTC TCC TGC GCA (SEQ ID NO: 7 - GCC TCCGGG TTC AGG TTC ACC bases 58-147) CDR 1  31              35 35a 35b PheAsn Asn Tyr Tyr Met Asp (SEQ ID NO: 8 - residues 50-56) TTC AAT AAC TACTAC ATG GAC (SEQ ID NO: 7 - bases 148-168) Framework 2 36              40 Trp Val Arg Gln Ala Pro Gly Gln (SEQ ID NO: 8 -                     49 Gly Leu Glu Trp Val Ser residues 57-70) TGG GTCCGC CAC GCa CCA GGG CAG (SEQ ID NO: 7 - GGG CTG GAG TGG GTC TCA bases169-210) CDR2  50      52 52A  53                          60 Arg IleSer Ser Ser Gly Asp Pro Thr Trp Tyr Ala Asp Ser Val CGT ATT AGT AGT AGTGGT GAT CCC ACA TGG TAC GCA GAC TCC GTG  65 Lys Gly (SEQ ID NO: 8 -residues 58-87) AAG GGC (SEQ ID NO: 7 - bases 211-261) Framework 3 66              70 Arg Phe Thr Ile Ser Arg Glu Asn Ala Asn Asn Thr         80 Leu Phe Leu AGA TTC ACC ATC TCC AGA GAG AAC GCC AAC AAC ACACTG TTT CTT      82 82a 82b 82c 83 Gln Met Asn Ser Leu Arg Ala Glu AspThr Ala Val  90 Tyr Tyr Cys CAA ATG AAC AGC CTG AGA GCT GAG GAC ACG GCTGTC TAT TAC TGT  94 Ala Ser (SEQ ID NO: 8 - residues 88-119) GCG AGC(SEQ ID NO: 7 - bases 262-357) CDR 3  95                 100 101 Leu ThrThr Gly Ser Asp Ser (SEQ ID NO: 8 - residues 120-126) TTG ACT ACA GGGTCT GAC TCC (SEQ ID NO: 7 - bases 358-378) Framework 4 103 113 Trp GlyGln Gly Val Leu Val Thr (SEQ ID NO: 8 - 110 Val Ser Ser residues127-137) TGG GGC CAG GGA GTC CTG GTC ACC (SEQ ID NO: 7 - GTC TCC TCAbases 379-411)3) Construction of Mammalian Expression Vectors

The heavy variable domain in N5KG4P was digested with Nhe I and Sal I,purified, and cloned into N5KG4P which contains the light chain variabledomain of 5E8. This plasmid was then digested with the restrictionendonucleases as previously described. This resulted in a vectorcontaining both the light and heavy variable domain of 5E8. This vectorwas named N5KG4P+5E8. The heavy and light variable domains of N5KG4P+5E8were then both inserted into the mammalian expression vector N5KG1 tocreate the N5KG1+5E8 vector.

4) Alteration of an Amino Acid in the Heavy Chain Variable Region ofPrimate Monoclonal Antibody 5E8 by Site Specific Mutagenesis andConstruction of Mammalian Expression Vectors

Based upon the sequence of 5E8 heavy variable domain, there is apotential glycosylation site of the immunoglobulin at asparagine codon75. This potential glycosylation site corresponds to a conservedasparagine-linked glycosylation motif having the following tripeptidesequence: (Asn)-(Any amino acid except proline)-(Serine or threonine).Therefore, a glycosylation mutant of 5E8, which would be unable to beglycosylated at this position because of modification of thisglycosylation motif, was generated by replacing the asparagine codon 75with a lysine (which is found in many human immunoglobulins at thisposition). Site specific mutagenesis was effected by the followingmethods.

A first PCR was done using N5KG4P+5E8 as a template and a 3′ primer(corresponding to codon 71 to 79) and which contains a mutation at codon75 (AAC changed to AAG, Primer MB1654 (SEQ ID NO: 39), and a 5′ primerat the beginning of the leader sequence (Primer MB1650) (SEQ ID NO: 36).(See PCR Primers Used for the Generation of a Glycosylation Mutant ofthe Heavy Chain Variable Region 5E8 set forth in Table 5 (SEQ ID NOs:36-39)).

A second PCR was performed on the same template by using a 5′ primer(corresponding to codon 71 to 79) containing the same mutation (PrimerMB1653) (SEQ ID NO: 38) and a 3′ primer from the end of framework 4(Primer MB1651) (SEQ ID NO: 37) (See PCR Primers Used for the Generationof a Glycosylation Mutant of the Heavy Chain Variable Region of 5E8 inTable 5 (SEQ. ID NOs: 36-39).)

These two PCR products were isolated and mixed in equal molar ratios. Athird independent PCR was then carried out by using the mixture of thefirst and second PCP products as a template with a 5′ primer used in thefirst PCR (MB1650) (SEQ ID NO: 36) and a 3′ primer used in the secondPCR (MP 1651) (SEQ ID NO: 37) (See PCP Primers Used for the Generationof a Glycosylation Mutant of the Heavy Chain Variable Region in Table 5(SEQ ID NOs: 36-39).) The PCR product obtained in third PCR was found tocontain the heavy variable domain coding region of 5E8 wherein theasparagine 75 had been changed to lysine.

The third PCR product was purified, digested with restrictionendonucleases Sal I and Nhe I, and cloned into the N5KG4P containing thelight variable domain only. The PCR product was sequenced to confirmthat it comprised the mutant heavy variable domain. This mammalianexpression plasmid was named N5KG4P+5E8N−.

To construct of N5KG1+5E8N−, both light and heavy variable domains fromN5KG4P−5E8N− were digested with Bgl II and BsiW I, and Nhe I and Sal Irespectively. The light variable domain was cloned first and then theheavy variable domain was inserted into the mammalian expression plasmidN5KG1, to create the plasmid N5KG1+5E8N−. TABLE 1 Primers for PCR of thekappa light chain variable domain of 5E8 NAME FAMILY Light chain Vk-early leader 5′ (Bgl II)                           −22 −21 −20 GE201 5′AT CAC AGA TCT CTC ACC ATG GAC ATG 1 −19 −18 −17 −16 −15 −14 AGGGTC     CCC GCT     CAG  3′ (SEQ ID NO: 9) GE200 5′ AT CAC AGA TCT CTCACC         ATG 2 AGG CTC     CCT GCT     CAG  3′ (SEQ ID NO: 10) GE2025′ AT CAC AGA TCT CTC ACC         ATG 3′3 GAA (A/G)CC CCA GC(T/G)CAG  3′ (SEQ ID NO: 11) GE203 5′ AT CAC AGA TCT CTC ACC         ATG 3′4GTG TTG     CAG ACC     CAG GTC (SEQ ID NO: 12) Light chain Vk-3′ primer(BsiW I)           113 112 111 110 109 108 GE204 5′ GG TGC AGC CAC CGTAGC TTT GAT 107 106    105 104 103 (C/T)TC CA(G/C) CTT 3′ (SEQ ID NO:13)

TABLE 2 Primers for PCR of the lambda light chain variable domain of 6G5NAME FAMILY Light chain V1-early leader 5′ (Bgl II) −20  −19  −18 744 5′AT CAC AGA TCT CTC ACC ATG (G/A)CC 1    −17   −16  −15TG(G/C)  TCC     CCT CT 3′ (SEQ ID NO: 14) 745 5′ AT CAC AGA TCT CTC ACCATG GCC 2 TGG  (A/G)CT C(T/C)G CT 3′ (SEQ ID NO: 15) 910 5′ AT CAC AGATCT CTC ACC ATG GC(A/C) 3 TGG   A(T/C)C    CCT CTC 3′ (SEQ ID NO: 16)Light chain V1-3′ primer (Avr II)             110 109 108 107 106 105104 926 5′(AC)10 CTT GGG CTG ACC TAG GAC GGT 3′ (SEQ ID NO: 17)

TABLE 3 Primers for PCR of the heavy chain variable domains from 6G5 and5E8 NAME Family Heavy chain-early leaders 5′ (Sal I) MB1503 5′ GCG ACTAAG TCG ACC (SEQ ID NO: 18) 1 −20 −19 −18 −17 −16 −15 ATG GAC TGG ACCTGG 3′ MB1502 5′ GCG ACT AAG TCG ACC (SEQ ID NO: 19) 2,4 ATG AAA CAC CTGTGG 3′ GE207 5′ GCG ACT AAG TCG ACC (SEQ ID NO: 20) 3 ATG GAG TTT GGGCTG AGC 3′ GE208 5′ GCG ACT AAG TCG ACC (SEQ ID NO: 21) 5 ATG GGG TCAACC GCC ATC 3′ GE209 5′ GCG ACT AAG TCG ACC (SEQ ID NO: 22) 6 ATG TCTGTC TCC TTC CTC 3′ Heavy chain-3′ primer (Nhe I) GE244 5′ GC CAG GGG GAAGAC (SEQ ID NO: 23) 120 119 118 117 116 CGA TGG GCC CTT GGT 115 114 113112 111 110 GCT AGC TGA GGA GAC GG 3′ GE210 5′ (SEQ ID NO: 24)  GA TGGGCC CTT GGT GCT AGC TGA GGA GAC GG 3′ ME1533 5′ (SEQ ID NO: 25)                    GGT GCT AGC TGA GGA GAC GGT                   109                  GAC 108 107 106 105 104 103 CAG GAC TCC CTG GCC CCA101 100 99 GAA GCC TAG 3′

TABLE 4 Sequencing Primers SpG 5′ AT TTA GGT GAC ACT ATA (SEQ ID NO: 26)primer 3′ M13 5′ GTT TTC CCA GTC ACG A (SEQ ID NO: 27) (−40) 3′ ForwardPrimer T7 5′ AT ATA CGA CTC ACT ATA (SEQ ID NO: 28) Pro- GGG 3′ moterPrimer GE 108 5′ CCG TCA GAT CGC CTG GAG (SEQ ID NO: 29) Primer ACG CCA3′ 377 5′ GCA GTT CCA GAT TTC AAC (SEQ ID NO: 30) Primer TG 3′ 607 5′CCA GGC CAC TGT CAC GGC (SEQ ID NO: 31) PRIMER TTC 3′ 266 5′ CAG AGC TGGGTA CGT CCT (SEQ ID NO: 32) PRIMER CA 3′ 268 5′ GCC CCC AGA GGT GCT CTT(SEQ ID NO: 33) PRIMER GG 3′ 876 5′ ACA CAG ACC CGT CGA CAT (SEQ ID NO:34) PRIMER GG 3′ 928 5′ GCT CTC GGA GGT GCT CCT (SEQ ID NO: 35) PRIMERGG 3′

TABLE 5 PCR Primers Used for the Generation of a Glycosylation Mutant ofthe Heavy Chain Variable Region of 5E8                Sal I   −20 MB1650 5′ ACA GAC CCG TCG ACC ATG (SEQ ID NO: 36) −19 −18 −17 −16 GAG TTTGGG CTG 3′                 Nhe I    118 117 116 115 114 113 MB 1651 5′CCC CTT GGT GCT AGC TGA (SEQ ID NO: 37) 112 111 110 GGA GAC GGT 3′        71  72  73  74  75 MB 1653 5′ AGA GAG AAC GCC AAG AAC (SEQ IDNO: 38)  76  77  78  79 ACA CTG TTT 3′         79  78  77  76  75 MB1654 5′ AAA CAG TGT GTT CTT GGC (SEQ ID NO: 39)  74  73  72  71 GTT CTCTCT 3′C) Expression of PRIMATIZED® Antibodies in Chinese Hamster Ovary Cells

A large scale plasmid DNA was purified using the WIZARD® Maxipreps DNAPurification System (Promega Catalog # A7421).

The purified DNA was digested with Ssp I and BspLU11 I, precipitatedwith ethanol once, and resuspended in sterile TE.

Purified, endonuclease restricted plasmid DNA was then introduced intoChinese hamster ovary (CHO) dihydrofolate reductase minus DG44 cellsusing electroporation. The electroporation technique used is describedbelow.

Approximately 1.6×10⁸ CHO cells were spun in an appropriate size sterileCorning tube for one minute at 1000 RPM. The media was removed and thecells were washed in fifteen milliliters of sterile ice cold SBS(sterile sucrose buffered solution is 272 mM sucrose, 7 mM sodiumphosphate pH 7.4, 1 mM MgCl₂) and spun for 5 minutes at 1000 RPM. TheSBS was removed and cells were suspended using fresh ice cold sterileSBS at a cell concentration of 1×10⁷ cells were per ml and left on icefor 15 minutes. The BTX 600 electroporator was turned on and preset at230 volts, with the maximum voltage knobs being set at 500volts/capacitance & resistance. The capacitance was set at 400microfaradays and the resistance was set at 13 ohms (setting R1).

Plasmid DNA (4 μg DNA or 2 μg DNA) and 0.4 ml of cells (4×10⁶ cells)were then placed in BTX 0.4 ml cuvettes (BTX Catalog # 620). The cellswere shocked by placing the cuvette into the BTX 600 stand and pressingthe automatic charge & pulse button. Approximately 20 separateelectroporations were performed with each mammalian expression plasmid.

After shocking., the cuvettes were left at ambient temperature forfifteen minutes. The cells and DNA were from each cuvette wereresuspended in 20 ml of CHO-SSFMII containing no hypoxanthine orthymidine (Gibco BRL Catalog # 31033-012) to which HT supplement (100×supplement is 10 mM sodium hypoxanthine, 1.6 mM thymidine Gibco BRLCatalog # 11067-014) had been added. The cells from a singleelectroporated cuvette were then plated into a 96 well plates (200μl/well) and placed into a 37° C. CO₂ incubator. Selection was startedtwo or three days later by changing the media to the above media withthe addition of 400 mg/ml of Geneticin® (G418, Gibco BRL Catalog #10131-019). The cells were grown at 37° C. and the cell media werechanged every 3-5 days. After sixteen days G418 resistant clonesappeared in the wells and the supernatant was assayed for antibodyexpression by ELISA. The highest expressing clones were then expandedindividually. Monoclonal antibodies were purified as described below.

Immunoglobulin ELISA

Plates (Immulon 2, Dynatech Laboratories, Inc. Catalog # 011-010-3455)are coated overnight at 4° C. with 200 ng unlabeled goat anti-human IgGantibody at 100 μl/well. This is effected using twenty milliliters ofunlabeled goat anti-human IgG/10 mls Coating Buffer/plate (BoehringerMannheim Ab Catalog # 605 400). (1:500 dilution of ˜1 mg/ml stock.) Thecoating buffer is then removed from the plates and dried using a papertowel. One hundred microliters of a dilution buffer/well is then added.

Antibody solutions and standards (100 ng/ml-2.5 ng/ml) are then added induplicate at 100 μl/well directly to the 100 ml dilution buffer. Theantibody solutions and standards are contained in dilution buffer. Theresultant solutions are then incubated for at least 1 hour at 37° C.

After incubation, the contents of each plate are removed and the platesare washed with tap water five times. The plates are then dried on apaper towel.

After drying of the plates, a second antibody is then added at 100μl/well. This second antibody is either goat anti-human Kappa-HRPO:added at 1/10,000 dilution or 1 μl Ab/10 mls dilution buffer/plate,available from Southern Biotechnology Associates, Inc. Catalog # 2060-05or a goat anti-human Lambda-HRPO; used at 1/20,000 dilution or 1 μlAb/20 mls dilution buffer/2 plates (available from SouthernBiotechnology Associates, Inc. Catalog # 2070-05).

The antibody and contents of the plate are allowed to incubate for onehour at 37° C. After incubation the contents of each plate are removed.The plates are again washed five time with tap water, and the washedplates are dried. To the dried plates is then added HRPO substrate (TMBMicrowell—two component) in an amount of 100 μl/well. (Five millilitersof TMB Peroxidase Substrate+five milliliters of Peroxidase SolutionB/plate (Kirdgaard and Perry Labs, TMB Microwell two component reagentsCatalog # 50-76-00).

The reaction is stopped by the addition of one hundred microliters of 2MH₂SO₄ to each well when the weakest standard (2.5 ng/ml) is visible overbackground. The optical density of wells in plates is then read using aplate reader, e.g., Molecular Devices Emax precision microplate readerset at wavelength: OD 450 and OPT2 (OD 540). ELISA BUFFERS CoatingBuffer Sodium Carbonate 0.8 gram/liter Sodium Bicarbonate 1.55gram/liter Adjust pH to 9.5 with ˜1 ml 1N HCl Dilution Buffer 0.5%Nonfat Dry Milk in PBS plus 0.01% Thimerosal (100 mg/L) (5 gm/L)Examples of ELISA values obtained using the above-described assay areset forth below. Standard OD 450 OD450 Average 100 ng/ml  0.805 0.8760.841 50 ng/ml 0.395 0.472 0.434 25 ng/ml 0.213 0.252 0.233 10 ng/ml0.089 0.105 0.097  5 ng/ml 0.054 0.055 0.055 2.5 ng/ml  0.031 0.0350.033  0 ng/ml 0.004 0.006 0.005

Standards in Dilution Buffer

Appropriate dilution of stock AB (sterile filtered in normal saline,protein determination by OD) to give 1 mg/ml

EXAMPLE

Chimeric monkey/human anti-CD4 (CE9.1) is 4.18 mg/ml

24 μl of above into 76 μl Dilution Buffer is 1 mg/ml

50 μl Stock Ab (1 mg/ml) into 450 μl Dilution Buffer (D.B.) is 100 μg/ml

50 μl of above mixture into 450 μl D.B. is 10 μg/ml

200 μl of above mixture into 1.8 mls D.B. is 1 μg/ml

1 ml of above mixture into 9 mls D.B. is 100 ng/ml *

5 ml of above mixture into 5 ml D.B. is 50 ng/ml *

5 ml of above mixture into 5 ml D.B. is 25 ng/ml *

4 ml of above mixture into 6 ml D.B. is 10 ng/ml *

5 ml of above mixture into 5 ml D.B. is 5 ng/ml *

5 ml of above mixture into 5 ml D.B. is 2.5 ng/ml *

* Standards used in the ELISA

Antibody Purification by Protein A

Procedure

The culture supernatant is centrifuged to remove cells and debris. Thecentrifuge is then filtered through a 0.2 μm filter. A protein Asepharose Fast flow column (recombinant protein A Sepharose Fast floe)(Pharmacia Biotech Catalog # 71-5.000-09) is then prepared andequilibrated using PBS (pH 7.4).

The supernatant is loaded on the column at an appropriate flow rate(e.g., 2 ml/min). After loading, the column is washed with 10 columnvolume of PBS (pH 7.4). The antibody is eluted from the column using anelution buffer (0.2M acetic acid, 0.1 M glycine pH 3.5) at 1 ml/min flowrate. One milliliter fractions/tube including 100 μl of Tris are thencollected. A spectrophotometer absorbance reading is then taken at 280nm. The antibody fractions are then collected and dialyzed against PBS(pH 7.9) overnight. The dialysate is then sterilized by filtrationthrough a 0.22 μm membrane and stored at −20° C.

Assay Results

The PRIMATIZED® human gamma-4 anti-human CD23 antibodies which aredescribed supra, were purified and assayed for induced IgE inhibitoryactivity in vitro. These results are contained in FIGS. 3 and 5. Thiswas effected using the in vitro IL-4 IgE assay described supra.

These assay results surprisingly indicated that both human gamma-4anti-human CD23 antibodies were not as active as the correspondingprimate anti-human CD23 antibodies, i.e., they did not significantlyinhibit induced IgE production in vitro.

However, because primate 5E8 and p5E8G4P have a potential asparaginelinked glycosylation site in the heavy chain variable region, theeffects of glycosylation at this site were investigated. (It was foundthat both these antibodies contain N-linked oligosaccharides at thissite. (Data not shown.)) Therefore, in order to prevent glycosylation,the asparagine in the glycosylation site was changed to a lysine inorder to eliminate carbohydrate addition. This mutated antibody wasnamed p5E8G4PN-. Assay results demonstrated that this antibody behavedidentically to p5E8G4P in the IL-4 IgE assay (see FIG. 3) and alsoexhibited an identical apparent affinity Kd for human CD23. (See FIG.4.) Therefore, these results indicated that the difference in IgEinhibition observed from the 5E8 gamma 4 PRIMATIZED® antibody incomparison to primate 5E8 antibody was not attributable to glycosylationdifferences.

The three primate antibodies (p5E8G4P, p5E8G4PN−, and p6G5G4P) were thenexpressed as human gamma-1 versions using substantially the samemethodology. All three human gamma-1 anti-human CD23 antibodies,respectively designated p5E8G1, p5E8G1N− and p6G5G1, were found to beactive in the in vitro IL-4/IgE′ assay (FIGS. 3 and 5).

p5E8G1 was found to be statistically more suppressive than p5E8G4P at aconcentration of 0.3 μg/ml (P[T,t] one tail+0.0055) and at 3 μg/ml(p[T<t] one tail+0.0019). In addition, p5E8G1N− is statistically moresuppressive than p5E8G4PN− at both 0.3 μg/ml (p[T<t] one tail+0.0392)and at 3 μg/ml (p[T<t] one tail+0.0310) (FIG. 3).

Similarly, p6G5G1 completely inhibited induced IgE production at 3mg/ml, while p6G5G4P did not. (These results are in FIG. 5).

Thus, these results suggested that an active Fc region, in particularthat of human gamma-1, is significant for induced IgE inhibition byanti-human CD23 antibodies.

Example 2

To confirm our hypothesis as to the involvement of the Fc effectorportion in IgE inhibition of anti-human CD23 antibodies, a third primateantibody, designated 2C8, also shown to inhibit IgE in in vitro) wasconverted to a F(ab′ )₂. IgE inhibitory activity was determined usingthe same IL-4/IgE assay described previously.

Materials

The following materials were used in this example.

ImmunoPure F(ab′)₂ Preparation Kit (Pierce Catalog # 44888)

-   -   digestion buffer: 20 mM sodium acetate buffer, pH 4.5    -   0.1 M citric acid, pH 3.0 (adjust pH with NaOH)    -   0.1% sodium azide in water    -   dialysis tubing; 50,000 MW cutoff (Spectra Por Catalog # 132        128)    -   shaking water bath capable of maintaining 37° C.    -   polystyrene culture tubes, 17×100 mm (Fisher Catalog #        14-956-6B)    -   BCA protein assay (Pierce Catalog # 23224)    -   Centricon-50 concentrators (Amicon Catalog # 4225).        Equilibration of Immobilized Pepsin

0.25 milliliters of the 50% slurry of Immobilized Pepsin is added to a17×100 mm test tube (0.125 ml of gel). Four milliliters of digestionbuffer are then added. The pepsin is then separated from the bufferusing the serum separator. The buffer is then discarded and the washprocedure repeated using another four milliliters of buffer. Theimmobilized pepsin is then resuspended in 0.5 ml of digestion buffer;

Preparation of Immobilized Protein A Column

Protein A AffinityPak® columns and ImmunoPure Binding and Elutionbuffers are brought to room temperature.

Preparation of 2C8 F(ab′)₂ Fragments

2C8 F(ab′)₂ fragments are prepared by methods well known in the antibodyart. The inventors elected to use a commercially available kit,ImmunoPure F(ab′)₂ Preparation Kit (Pierce Catalog # 44888), using themanufacturer's protocols.

Ten milligrams of lyophilized 2C8 antibody were dissolved in onemilliliter of a digestion buffer (20 mM sodium acetate buffer, pH 4.5).One milliliter of the antibody containing sample was than added to atube containing immobilized pepsin.

The antibody and immobilized pepsin were then incubated for four hoursin a high speed shaker water bath at 37° C. (at high speed), taking careto maintain the mixing constant during the incubation.

The resultant solubilized F(ab′)₂ and Fc fragments and the undigestedIgGs were then recovered from the immobilized. pepsin gel using a serumseparator. The crude digest is then decanted into a clean tube.

In order to enhance recovery of F(ab′)₂ fragments, the immobilizedpepsin desirably is then washed with 1.5 of milliliters of theImmunoPure IgG binding buffer. The wash is then added to the crudedigest.

The antibody fragments were then recovered using a protein A column.This is effected by opening an immobilized protein A column. Care istaken to avoid air bubbles from being drawn into the gel. The storagesolution (which contains 0.02% sodium azide) is discarded.

The immobilized protein A column was then equilibrated using twelvemilliliters of binding buffer (contained in ImmunoPure Preparation Kit).The column was then transferred to a 17×100 mn test tube contained inthe kit (labeled “F(ab′)₂”) to collect eluate.

Three milliliters of the crude digest was then applied to a column andare allowed to flow completely into the gel. The use of AffinityPak™columns is desirable as these columns stop flowing automatically whenthe level reaches the top frit.

The column is then washed using six milliliters of binding buffer. Theeluate which contains F(ab′ )₂ fragments was then collected. This eluatealso contains small Fc fragments that can no longer bind protein A(which are not bound to the Protein A column). However, the substantialportion thereof was eliminated by dialysis.

Dialysis was effected by taking the F(ab′)₂ containing eluate anddialyzing the eluate against pH 7.4 phosphate buffered saline, usingdialysis tubing with a molecular weight cut-off of 50,000 so as toeliminate the small Fc fragment containments (Spectra Pur. Catalog # 132128).

This resulted in a F(ab′)₂ fraction having an optical density of 280 nmof 0.707 (6 ml). After dialysis and concentration with Centricon-50concentrators (Amicon Catalog # 4225), the 2C8 F(ab′)₂ product wasassayed for protein content using a BCA protein assay (Pierce Catalog #23224). The protein content was found to be 3.76 mg per milliliter.

The 2C8 F(ab)₂′S were assayed for IgE inhibitory activity and were foundto be substantially incapable of inhibiting IgE production in the samein vitro assays described previously. These results are contained inFIG. 6. In fact, the F(ab′)₂ was found to antagonize the suppressiveeffects of induced IgE on the monoclonal antibody 2C8. These results arein FIG. 7.

Example 3

The PRIMATIZED® gamma 1 and gamma 4P versions of primate monoclonal 6G5were both evaluated for their effect on inhibition of induced IgEproduction in vivo in the SCID mouse model described previously.p5E8G1N− was found to be as efficient as primate 5E8 in inhibitinginduced IgE. (See FIGS. 8 and 9). While neither provide 6G5 nor theprimatized p6G5G4P were effective at inhibiting induced IgE in vivo,primatized p6G5G1 inhibited induced IgE production. (See FIGS. 9 and10.) These results further substantiate our conclusion that an active Fcregion is significant to the ability of an anti-human CD23 antibody toeffectively inhibit induced IgE production.

Utility

The subject anti-human CD23 antibodies which comprise human gamma-1constant domains, because of their ability to effectively inhibit IgEproduction, are effective in treating any disease wherein inhibition ofIgE production is therapeutically desirable. Such diseases include byway of example allergic diseases, autoimmune diseases and inflammatorydisease.

Specific conditions which are potentially treatable by administration ofthe subject anti-CD23 human gamma-1 constant domain containingantibodies include the following:

Allergic bronchopulmonary aspergillosis; Allergic rhinitis andconjunctivitis autoimmune hemolytic anemia; Acanthosis nigricans;Allergic contact dermatitis; Addison's disease; Atopic dermatitis;Alopecia areata; Alopecia universalis; Amyloidosis; Anaphylactoidpurpura; Anaphylactoid reaction; Aplastic anemia; Angioedema,hereditary; Angioedema, idiopathic; Ankylosing spondylitis; Arteritis,cranial; Arteritis, giant cell; Arteritis, Takayasu's; Arteritis,temporal; Asthma; Ataxia-telangiectasia; Autoimmune oophoritis;Autoimmune orchitis; Autoimmune polyendocrine failure; Behcet's disease;Berger's disease; Buerger's disease; bronchitis; Bullous pemphigus;Candidiasis, chronic mucocutaneous; Caplan's syndrome; Post-myocardialinfarction syndrome; Post-pericardiotomy syndrome; Carditis; Celiacsprue; Chagas's disease; Chediak-Higashi syndrome; Churg-Straussdisease; Cogan's syndrome; Cold agglutinin disease; CREST syndrome;Crohn's disease; Cryoglobulinemia; Cryptogenic fibrosing alveolitis;Dermatitis herpetifomis; Dermatomyositis; Diabetes mellitus;Diamond-Blackfan syndrome; DiGeorge syndrome; Discoid lupuserythematosus; Eosinophilic fasciitis; Episcleritis; Drythema elevatumdiutinum; Erythema marginatum; Erythema multiforme; Erythema nodosum;Familial Mediterranean fever; Felty's syndrome; Fibrosis pulmonary;Glomerulonephritis, anaphylactoid; Glomerulonephritis, autoimmune;Glomerulonephritis, post-streptococcal; Glomerulonephritis,post-transplantation; Glomerulopathy, membranous; Goodpasture'ssyndrome; Graft-vs.-host disease; Granulocytopenia, immune-mediated;Granuloma annulare; Granulomatosis, allergic; Granulomatous myositis;Grave's disease; Hashimoto's thyroiditis; Hemolytic disease of thenewborn; Hemochromatosis, idiopathic; Henoch-Schoenlein purpura;Hepatitis, chronic active and chronic progressive; Histiocytosis_X;Hypereosinophilic syndrome; Idiopathic thrombocytopenic purpura; Job'ssyndrome; Juvenile dermatomyositis; Juvenile rheumatoid arthritis(Juvenile chronic arthritis); Kawasaki's disease; Keratitis;Keratoconjunctivitis sicca; Landry-Guillain-Barre-Strohl syndrome;Leprosy, lepromatous; Loeffler's syndrome; lupus; Lyell's syndrome; Lymedisease; Lymphomatoid granulomatosis; Mastocytosis, systemic; Mixedconnective tissue disease; Mononeuritis multiplex; Muckle-Wellssyndrome; Mucocutaneous lymph node syndrome; Mucocutaneous lymph nodesyndrome; Multicentric reticulohistiocytosis; Multiple sclerosis;Myasthenia gravis; Mycosis fungoides; Necrotizing vasculitis, systemic;Nephrotic syndrome; Overlap syndrome; Panniculitis; Paroxysmal coldhemoglobinuria; Paroxysmal nocturnal hemoglobinuria; Pemphigoid;Pemphigus; Pemphigus erythematosus; Pemphigus foliaceus; Pemphigusvulgaris; Pigeon breeder's disease; Pneumonitis, hypersensitivity;Polyarteritis nodosa; Polymyalgia rheumatic; Polymyositis; Polyneuritis,idiopathic; Portuguese familial polyneuropathies;Pre-eclampsia/eclampsia; Primary biliary cirrhosis; Progressive systemicsclerosis (Scleroderma); Psoriasis; Psoriatic arthritis; Pulmonaryalveolar proteinosis; Pulmonary fibrosis, Raynaud's phenomenon/syndrome;Reidel's thyroiditis; Reiter's syndrome, Relapsing polychrondritis;Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Scleritis;Sclerosing cholangitis; Serum sickness; Sezary syndrome; Sjogren'ssyndrome; Stevens-Johnson syndrome; Still's disease; Subacute sclerosingpanencephalitis; Sympathetic ophthalmia; Systemic lupus erythematosus;Transplant rejection; Ulcerative colitis; Undifferentiated connectivetissue disease; Urticaria, chronic; Urticaria, cold; Uveitis; Vitiligo;Weber-Christian disease; Wegener's granulomatosis; Wiskott-Aldrichsyndrome.

Of these, the preferred indications treatable or presentable byadministration of anti-CD23 antibodies include allergic rhinitis andconjunctivitis, atopic dermatitis; eczema; Job's syndrome, asthma; andallergic conditions; chronic inflammatory diseases and conditions.

The amount of antibody useful to produce a therapeutic effect can bedetermined by standard techniques well known to those of ordinary skillin the art. The antibodies will generally be provided by standardtechnique within a pharmaceutically acceptable buffer, and may beadministered by any desired route. Because of the efficacy of thepresently claimed antibodies and their tolerance by humans it ispossible to administer these antibodies repetitively in order to combatvarious diseases or disease states within a human.

One skilled in the art would be able, by routine experimentation, todetermine what an effective, non-toxic amount of antibody would be forthe purpose of effecting allergic diseases and inflammatory conditions.Generally, however, an effective dosage will be in the range of about0.05 to 100 milligrams per kilogram body weight per day.

The antibodies of the invention may be administered to a human or otheranimal in accordance with the aforementioned methods of treatment in anamount sufficient to produce such effect to a therapeutic orprophylactic degree. Such antibodies of the invention can beadministered to such human or other animal in a conventional dosage formprepared by combining the antibody of the invention with a conventionalpharmaceutically acceptable carrier or diluent according to knowntechniques. It will be recognized by one of skill in the art that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.

The route of administration of the antibody of the invention may beoral, parenteral, by inhalation or topical. The term parenteral as usedherein includes intravenous, intramuscular, subcutaneous, rectal,vaginal or intraperitoneal administration. The intravenous form ofparenteral administration is generally preferred.

The daily parenteral and oral dosage regimens for employing compounds ofthe invention to prophylactically or therapeutically induceimmunosuppression will generally be in the range of about 0.05 to 100,but preferably about 0.5 to 10, milligrams per kilogram body weight perday.

The antibody of the invention may also be administered by inhalation. By“inhalation” is meant intranasal and oral inhalation administration.Appropriate dosage forms for such administration, such as an aerosolformulation or a metered dose inhaler, may be prepared by conventionaltechniques. The preferred dosage amount of a compound of the inventionto be employed is generally within the range of about 10 to 100milligrams.

The antibody of the invention may also be administered topically. Bytopical administration is meant non-systemic administration and includesthe application of an antibody (or fragment thereof) compound of theinvention externally to the epidermis, to the buccal cavity andinstillation of such an antibody into the ear, eye and nose, and whereit does not significantly enter the blood stream. By systemicadministration is meant oral, intravenous, intraperitoneal andintramuscular administration. The amount of an antibody required fortherapeutic or prophylactic effect will, of course, vary with theantibody chosen, the nature and severity of the condition being treatedand the animal undergoing treatment, and is ultimately at the discretionof the physician. A suitable topical dose of an antibody of theinvention will generally be within the range of about 1 to 100milligrams per kilogram body weight daily.

Formulations

While it is possible for an antibody or fragment thereof to beadministered alone, it is preferable to present it as a pharmaceuticalformulation. The active ingredient may comprise, for topicaladministration, from 0.001% to 10% w/w, e.g., from 1% to 2% by weight ofthe formulation, although it may comprise as much as 10% w/w butpreferably not in excess of 5% w/w and more preferably from 0.1% to 1%w/w of the formulation.

The topical formulations of the present invention, comprise an activeingredient together with one or more acceptable carrier(s) therefor andoptionally any other therapeutic ingredients(s). The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for′ penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or fungicidal agent and/or any other suitable preservative, andpreferably including a surface active agent. The resulting solution maythen be clarified by filtration, transferred to a suitable containerwhich is then sealed and sterilized by autoclaving or maintaining at90°-100° C. for half an hour. Alternatively, the solution may besterilized by filtration and transferred to the container by an aseptictechnique. Examples of bactericidal and fungicidal agents suitable forinclusion in the drops are phenylmercuric nitrate or acetate (0.002%),benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).Suitable solvents for the preparation of an oily solution includeglycerol, diluted alcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy basis. The basis may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives, or a fattyacid such as stearic or oleic acid together with an alcohol such aspropylene glycol or macrogels. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurface active such as sorbitan esters or polyoxyethylene derivativesthereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of an antibody or fragmentthereof of the invention will be determined by the nature and extent ofthe condition being treated, the form, route and site of administration,and the particular animal being treated, and that such optimums can bedetermined by conventional techniques. It will also be appreciated byone of skill in the art that the optimal course of treatment, i.e., thenumber of doses of an antibody or fragment thereof of the inventiongiven per day for a defined number of days, can be ascertained by thoseskilled in the art using conventional course of treatment determinationtests.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following are, therefore, to be construed asmerely illustrative examples and not a limitation of the scope of thepresent invention in any way.

Capsule Composition

A pharmaceutical composition of this invention in the form of a capsuleis prepared by filling a standard two-piece hard gelatin capsule with 50mg. of an antibody or fragment thereof of the invention, in powderedform, 100 mg. of lactose, 32 mg. of talc and 8 mg. of magnesiumstearate.

Injectable Parenteral Composition

A pharmaceutical composition of this invention in a form suitable foradministration by injection is prepared by stirring 1.5 k by weight ofan antibody or fragment thereof of the invention in 10 k by volumepropylene glycol and water. The solution is sterilized by filtration.

Ointment Composition

Antibody or fragment thereof of the invention 10 g.

White soft paraffin to 100.0 g.

The antibody or fragment thereof of the invention is dispersed in asmall volume of the vehicle to produce a smooth, homogeneous product.Collapsible metal tubes are then filled with the dispersion.

Topical Cream Composition

Antibody or fragment thereof of the invention 1.0 g.

Polawax GP 200 20.0 g.

Lanolin Anhydrous 2.0 g.

White Beeswax 2.5 g.

Methyl hydroxybenzoate 0.1 g.

Distilled Water to 100.0 g.

The polawax, beeswax and lanolin are heated together at 60° C. Asolution of methyl hydroxybenzoate is added and homogenization isachieved using high speed stirring. The temperature is then allowed tofall to SOOC. The antibody or fragment thereof of the invention is thenadded and dispersed throughout, and the composition is allowed to coolwith slow speed stirring.

Topical Lotion Composition

Antibody or fragment thereof of the invention 1.0 g.

Sorbitan Monolaurate 0.6 g. Polysorbate 20 0.6 g.

Cetostearyl Alcohol 1.2 g. Glycerin 6.0 g.

Methyl Hydroxybenzoate 0.2 g.

Purified Water B.P. to 100.00 ml. (B.P.=British Pharmacopeia)

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml. of thewater at 75° C. The sorbitan monolaurate, polysorbate 20 and cetostearylalcohol are melted together at 75° C. and added to the aqueous solution.The resulting emulsion is homogenized, allowed to cool with continuousstirring and the antibody or fragment thereof of the invention is addedas a suspension in the remaining water. The whole suspension is stirreduntil homogenized.

Eye Drop Composition

Antibody or fragment thereof of the invention 0.5 g.

Methyl Hydroxybenzoate 0.01 g.

Propyl Hydroxybenzoate 0.04 g.

Purified Water B.P. to 100.00 ml.

The methyl and propyl hydroxybenzoates are dissolved in 70 ml. purifiedwater at 75° C. and the resulting solution is allowed to cool. Theantibody or fragment thereof of the invention is then added, and thesolution is sterilized by filtration through a membrane filter (0.022 Ampore size), and packed aseptically into suitable sterile containers.

Composition for Administration by Inhalation

For an aerosol container with a capacity of 15-20 ml: mix 10 mg. of anantibody or fragment thereof of the invention with 0.2-0.5 k of alubricating agent, such as polysorbate 85 or oleic acid, and dispersesuch mixture in a propellant, such as freon, preferably in a combinationof (1,2 dichlorotetrafluoroethane) and difluorochloromethane and putinto an appropriate aerosol container adapted for either intranasal ororal inhalation administration. Composition for Administration byInhalation For an aerosol container with a capacity of 15-20 ml:dissolve 10 mg. of an antibody or fragment thereof of the invention inethanol (6-8 ml.), add 0.1-0.2 k of a lubricating agent, such aspolysorbate 85 or oleic acid; and disperse such in a propellant, such asfreon, preferably in combination of (1-2 dichlorotetrafluoroethane) anddifluorochloromethane, and put into an appropriate aerosol containeradapted for either intranasal or oral inhalation administration.

Parenteral Administrable Antibody Compositions

The antibodies and pharmaceutical compositions of the invention areparticularly useful for parenteral administration, i.e., subcutaneously,intramuscularly or intravenously. The compositions for parenteraladministration will commonly comprise a solution of an antibody of theinvention or a cocktail thereof dissolved in an acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers may beemployed, e.g., water, buffered water, 0.4 k saline (normal saline),0.3% glycine, and the like. The use of normal saline is preferred. Thesesolutions are sterile and generally free of particulate matter. Thesesolutions may be sterilized by conventional, well-known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, etc. The concentration of theantibody or fragment thereof of the invention in such pharmaceuticalformulation can vary widely. Such concentrations will be selectedprimarily based on fluid volumes, viscosities, etc., according to theparticular mode of administration selected. Generally suitableintravenous concentrations range from about one to one hundredmilligrams per milliliter.

Thus, a pharmaceutical composition of the invention for intravenousinjection could comprise 10 mL normal saline containing 40-50 mg of ananti-human CD23 antibody of the invention. Methods for preparingparenterally administrable compositions are well-known or will beapparent to those skilled in the art, and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa., hereby incorporated by referenceherein.

The antibodies of the invention can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immune globulins andart-known lyophilization and reconstitution techniques can be employed.

Depending on the intended result, the pharmaceutical composition of theinvention can be administered for prophylactic and/or therapeutictreatments. In therapeutic application, compositions are administered toa patient already suffering from a disease, in an amount sufficient tocure or at least partially arrest the disease and its complications. Inprophylactic applications, compositions containing the presentantibodies or a cocktail thereof are administered to a patient notalready in a disease state to enhance the patient's resistance.

Single or multiple administrations of the pharmaceutical compositionscan be carried out with dose levels and pattern being selected by thetreating physician. In any event, the pharmaceutical composition of theinvention should provide a quantity of the subject anti-CD23 antibodiessufficient to effectively treat the patient.

It should also be noted that the antibodies of this invention may beused for the design and synthesis of either peptide or non-peptidecompounds (mimetics) which would be useful in the same therapy as theantibody. See, e.g., Saragovi et al., Science, 253:792-795 (1991).

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without diverting fromthe scope of the invention. Accordingly, the invention is not limited bythe appended claims.

1-55. (canceled)
 56. A method of inhibiting IgE in a human subjectcomprising administering to the human subject a therapeuticallyeffective amount of an anti-human CD23 antibody that (a) binds humanCD23; (b) comprises the complementarity-determining regions CDR1, CDR2,and CDR3 of the light and heavy chains of antibody 6G5, wherein CDR1,CDR2, and CDR3 of the light chain of antibody 6G5 consist of amino acids23-36, 52-58, and 91-100, respectively, of SEQ ID NO:2; and CDR1, CDR2,and CDR3 of the heavy chain of antibody 6G5 consist of amino acids31-36, 51-67, and 100-111, respectively, of SEQ ID NO:4; and (c)inhibits IL-4-induced IgE production by B-cells.
 57. The method of claim56, wherein the anti-human CD23 antibody comprises a light chainvariable region of amino acids 1-111 of SEQ ID NO:2 and a heavy chainvariable region of amino acids 1-122 of SEQ ID NO:4.
 58. The method ofclaim 56, wherein the anti-human CD23 antibody comprises a human gamma-Iconstant region.
 59. The method of claim 56, wherein the anti-human CD23antibody comprises a human gamma-3 constant region.
 60. The method ofclaim 56, wherein the anti-human CD23 antibody binds CD23 with a bindingaffinity ranging from 0.01 nM to 1000 nM.
 61. The method of claim 60,wherein the anti-human CD23 antibody binds CD23 with a binding affinityranging from 5 nM to 1000 nM.
 62. The method of claim 61, wherein theanti-human CD23 antibody binds CD23 with a binding affinity ranging from100 nM to 1000 nM.
 63. The method of claim 56, wherein the anti-humanCD23 antibody is administered parenterally by subcutaneous,intravascular, intramuscular, rectal, vaginal, or intraperitonealadministration.
 64. The method of claim 63, wherein the anti-human CD23antibody is administered by subcutaneous administration.
 65. The methodof claim 56, wherein the anti-human CD23 antibody is lyophilized forstorage and reconstituted prior to administration.
 66. The method ofclaim 56, wherein IgE is inhibited in a subject having an autoimmunedisease.
 67. The method of claim 66, wherein the autoimmune disease isrheumatoid arthritis.
 68. A method of inhibiting IgE in a human subjectcomprising administering to the human subject an effective amount of ananti-human CD23 antibody an anti-CD23 antibody that (a) binds humanCD23; (b) comprises the complementarity-determining regions CDR1, CDR2,and CDR3 of the light and heavy chains of antibody 5E8, wherein CDR1,CDR2, and CDR3 of the light chain of antibody 5E8 consist of amino acids24-34, 50-56, and 89-97, respectively, of SEQ ID NO:6; and CDR1, CDR2,and CDR3 of the heavy chain of antibody 5E8 consist of amino acids31-37, 52-68, and 101-107, respectively, of SEQ ID NO:8; and (c)inhibits IL-4-induced IgE production by B-cells.
 69. The method of claim68, wherein the anti-human CD23 antibody comprises a light chainvariable region of amino acids 1-107 of SEQ ID NO:6, and a heavy chainvariable region amino acids 1- 118 of SEQ ID NO:8, with the exceptionthat the asparagine residue at position +78 of SEQ ID NO:8 is replacedwith a lysine residue.
 70. The method of claim 68, wherein theanti-human CD23 antibody comprises a human gamma-I constant region. 71.The method of claim 68, wherein the anti-human CD23 antibody comprises ahuman gamma-3 constant region.
 72. The method of claim 68, wherein theanti-human CD23 antibody binds CD23 with a binding affinity ranging from0.01 nM to 1000 nM.
 73. The method of claim 72, wherein the anti-humanCD23 antibody binds CD23 with a binding affinity ranging from 5 nM to1000 nM.
 74. The method of claim 73, wherein the anti-human CD23antibody binds CD23 with a binding affinity ranging from 100 nM to 1000nM.
 75. The method of claim 68, wherein the anti-human CD23 antibody isadministered parenterally by subcutaneous, intravascular, intramuscular,rectal, vaginal, or intraperitoneal administration.
 76. The method ofclaim 75, wherein the anti-human CD23 antibody is administered bysubcutaneous administration.
 77. The method of claim 68, wherein theanti-human CD23 antibody is lyophilized for storage and reconstitutedprior to administration.
 78. The method of claim 68, wherein IgE isinhibited in a subject having an autoimmune disease.
 79. The method ofclaim 78, wherein the autoimmune disease is rheumatoid arthritis.